Multivalent synthetic nanocarrier vaccines

ABSTRACT

The invention relates, at least in part, to compositions comprising populations of synthetic nanocarriers that comprise different sets of antigens as well as related methods.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119 of U.S.provisional applications 61/348,713, filed May 26, 2010, 61/348,717,filed May 26, 2010, 61/348,728, filed May 26, 2010, and 61/358,635,filed Jun. 25, 2010, the entire contents of each of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

Multivalent vaccines are a useful way of generating an immune responseto certain foreign substances that otherwise would not be desirablyrobust. For instance, vaccinating against multiple strains of a virusmay provide more robust cross-protection against multiple strains ofthat virus as compared to vaccinating using a monovalent vaccine.

However, current multivalent vaccines and methods of making them needimprovement. For instance, current approaches of conjugating antigens toprotein carriers are complex and provide for low yields. Additionally,new techniques often need to be developed to conjugate new antigens tocarrier proteins because conventional techniques can be unsuccessful dueto the relative fragility of conventional carrier proteins.

What is needed are compositions and methods that provide for improvedmultivalent vaccines.

SUMMARY OF THE INVENTION

In one aspect, a composition comprising a dosage form comprising a firstpopulation of synthetic nanocarriers that comprise a first set ofsurface antigens; a second population of synthetic nanocarriers thatcomprise a second set of surface antigens; and a pharmaceuticallyacceptable excipient, wherein the first set of surface antigens and thesecond set of surface antigens are structurally different is provided.

In another aspect, a composition comprising a dosage form comprising afirst population of synthetic nanocarriers that comprise a first set ofsurface antigens; a second population of synthetic nanocarriers thatcomprise a second set of surface antigens; and a pharmaceuticallyacceptable excipient, wherein the first set of surface antigens and thesecond set of surface antigens are immunologically different is alsoprovided.

In yet another aspect, a composition comprising a dosage form comprisinga first synthetic nanocarrier means for presenting a first set ofsurface antigens; a second synthetic nanocarrier means for presenting asecond set of surface antigens; and a pharmaceutically acceptableexcipient, wherein the first set of surface antigens and the second setof surface antigens are structurally different is also provided.

In still another aspect, a composition comprising a dosage formcomprising a first synthetic nanocarrier means for presenting a firstset of surface antigens; a second synthetic nanocarrier means forpresenting a second set of surface antigens; and a pharmaceuticallyacceptable excipient, wherein the first set of surface antigens and thesecond set of surface antigens are immunologically different isprovided.

In one embodiment of any of the compositions provided, the first set ofsurface antigens comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more typesof antigens. In another embodiment of any of the compositions provided,the second set of surface antigens comprises 2, 3, 4, 5, 6, 7, 8, 9, 10or more types of antigens.

In yet another embodiment of any of the compositions provided, the firstset of surface antigens comprise antigens obtained or derived from afirst infectious genus and the second set of surface antigens compriseantigens obtained or derived from a second infectious genus. In oneembodiment, the first infectious genus and the second infectious genusare the same. In still another embodiment of any of the compositionsprovided, the first set of surface antigens comprise antigens obtainedor derived from a first infectious species and the second set of surfaceantigens comprise antigens obtained or derived from of a secondinfectious species. In one embodiment, the first infectious species andthe second infectious species are the same. In a further embodiment, thefirst set of surface antigens comprise antigens obtained or derived froma first infectious strain and the second set of surface antigenscomprise antigens obtained or derived from a second infectious strain.In one embodiment, the first infectious strain and second infectiousstrain are the same.

In another embodiment of any of the compositions provided, the first setof surface antigens and/or second set of surface antigens compriseantigens that are obtained or derived from a virus of the Adenoviridae,Picornaviridae, Herpesviridae, Hepadnaviridae, Flaviviridae,Retroviridae, Orthomyxoviridae, Paramyxoviridae, Papillomaviridae,Rhabdoviridae, Togaviridae or Paroviridae family. In one embodiment, thefirst set of surface antigens and/or second set of surface antigenscomprise antigens that are obtained or derived from adenovirus,coxsackievirus, hepatitis A virus, poliovirus, Rhinovirus, Herpessimplex virus, Varicella-zoster virus, Epstein-barr virus, Humancytomegalovirus, Human herpesvirus, Hepatitis B virus, Hepatitis Cvirus, yellow fever virus, dengue virus, West Nile virus, HIV, Influenzavirus, Measles virus, Mumps virus, Parainfluenza virus, Respiratorysyncytial virus, Human metapneumovirus, Human papillomavirus, Rabiesvirus, Rubella virus, Human bocarivus or Parvovirus B19. In anotherembodiment, the first set of surface antigens and/or second set ofsurface antigens comprise antigens that are obtained or derived from VI,VII, E1A, E3-19K, 52K, VP1, surface antigen, 3A protein, capsid protein,nucleocapsid, surface projection, transmembrane proteins, UL6, UL18,UL35, UL38, UL19, early antigen, capsid antigen, Pp65, gB, p52, latentnuclear antigen-1, NS3, envelope protein, envelope protein E2 domain,gp120, p24, lipopeptides Gag (17-35), Gag (253-284), Nef (66-97), Nef(116-145), Pol (325-355), neuraminidase, nucleocapsid protein, matrixprotein, phosphoprotein, fusion protein, hemagglutinin,hemagglutinin-neuraminidase, glycoprotein, E6, E7, envelope lipoproteinor non-structural protein (NS).

In still another embodiment of any of the compositions provided, thefirst set of surface antigens and/or second set of surface antigenscomprise antigens that are obtained or derived from a bacteria of theBordetella, Borrelia, Brucella, Campylobacter, Chlamydia andChlamydophila, Clostridium, Corynebacterium, Enterococcus, Escherichia,Francisella, Haemophilus, Helicobacter, Legionella, Leptospira,Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia,Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema Vibrio orYersinia genus. In one embodiment, the first set of surface antigensand/or second set of surface antigens comprise antigens that areobtained or derived from Bordetella pertussis, Borrelia burgdorferi,Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis,Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis,Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile,Clostridium perfringens, Clostridium tetani, Corynebacteriumdiphtheriae, Enterococcus faecalis, Enterococcus faecium, Escherichiacoli, Francisella tularensis, Haemophilus influenzae, Helicobacterpylori, Legionella pneumophila, Leptospira interrogans, Listeriamonocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis,Mycobacterium ulcerans, Mycoplasma pneumoniae, Neisseria gonorrhoeae,Neisseria meningitides, Pseudomonas aeruginosa, Rickettsia rickettsii,Salmonella typhi, Salmonella typhimurium, Shigella sonnei,Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcussaprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae,Streptococcus pyogenes, Treponema pallidum, Vibrio cholerae or Yersiniapestis. In another embodiment, the first set of surface antigens and/orsecond set of surface antigens comprise antigens that are obtained orderived from pertussis toxin (PT), filamentous hemagglutinin (FHA),pertactin (PRN), fimbriae (FIM 2/3), VlsE; DbpA, OspA, Hia, PrpA, MltA,L7/L12, D15, 0187, VirJ, Mdh, AfuA, L7/L12, out membrane protein, LPS,antigen type A, antigen type B, antigen type C, antigen type D, antigentype E, FliC, FliD, Cwp84, alpha-toxin, theta-toxin, fructose1,6-biphosphate-aldolase (FBA), glyceraldehydes-3-phosphatedehydrogenase (GPD), pyruvate:ferredoxin oxidoreductase (PFOR),elongation factor-G (EF-G), hypothetical protein (HP), T toxin, Toxoidantigen, capsular polysaccharide, Protein D, Mip, nucleoprotein (NP),RD1, PE35, PPE68, EsxA, EsxB, RD9, EsxV, Hsp70, lipopolysaccharide,surface antigen, Sp1, Sp2, Sp3, Glycerophosphodiester Phosphodiesterase,outer membrane protein, chaperone-usher protein, capsular protein (F1)or V protein.

In yet another embodiment of any of the compositions provided, the firstset of surface antigens and/or second set of surface antigens compriseantigens that are obtained or derived from a fungus of the Candida,Aspergillus, Cryptococcus, Histoplasma, Pneumocystis or Stachybotrysgenus. In one embodiment, the first set of surface antigens and/orsecond set of surface antigens comprise antigens that are obtained orderived from C. albicans, Aspergillus fumigatus, Aspergillus flavus,Cryptococcus neoformans, Cryptococcus laurentii, Cryptococcus albidus,Cryptococcus gattii, Histoplasma capsulatum, Pneumocystis jirovecii orStachybotrys chartarum. In another embodiment, the first set of surfaceantigens and/or second set of surface antigens comprise antigens thatare obtained or derived from surface antigen, capsular glycoprotein,Yps3P, Hsp60, Major surface protein, MsgC1, MsgC3, MsgC8, MsgC9 orSchS34.

In still another embodiment of any of the compositions provided, thefirst set of surface antigens and/or second set of surface antigenscomprise antigens that are obtained or derived from any of theinfectious agents, viruses, bacteria, proteins, peptides, polypeptides,small molecules, polysaccharides or oligosaccharides provided herein.

In a further embodiment of any of the compositions provided, the firstset of surface antigens and second set of surface antigens compriseantigens obtained or derived from an abused or addictive substance. Inone embodiment, the abused or addictive substance is cocaine ornicotine.

In yet a further embodiment of any of the compositions provided, thefirst set of surface antigens and the second set of surface antigenscomprise the same surface antigens, and wherein at least one antigen ofthe first set of surface antigens is presented in a differentorientation than as presented in the second set of surface antigens.

In still a further embodiment of any of the compositions provided, thefirst set of surface antigens and the second set of surface antigenscomprise the same surface antigens, and wherein at least one antigen ofthe first set of surface antigens is presented in a differentconformation than as presented in the second set of surface antigens.

In another embodiment of any of the compositions provided, the molecularstructure of the first set of surface antigens and the second set ofsurface antigens are different.

In yet another embodiment of any of the compositions provided, the firstset of surface antigens and/or the second set of surface antigenscomprise surface antigens with a molecular weight of less than 10,000Da.

In still another embodiment of any of the compositions provided, thefirst set of surface antigens and/or the second set of surface antigenscomprise surface antigens that comprise peptides, proteins,oligosaccharides, polysaccharides and/or small molecules.

In a further embodiment of any of the compositions provided, at leastone surface antigen of the first set of surface antigens and/or at leastone surface antigen of the second set of surface antigens has amolecular weight of less than 10,000 Da.

In a yet a further embodiment of any of the compositions provided, thefirst set of surface antigens comprises surface antigens comprisingpeptides, and the second set of surface antigens comprises surfaceantigens with a molecular weight of less than 10,000 Da.

In still a further embodiment of any of the compositions provided, thefirst set of surface antigens comprises surface antigens comprisingpeptides, and the second set of surface antigens comprises surfaceantigens comprising peptides, proteins, oligosaccharides,polysaccharides and/or small molecules. In one embodiment, at least onesurface antigen of the second set of surface antigens has a molecularweight of less than 10,000 Da.

In another embodiment of any of the compositions provided, the first setof surface antigens comprises surface antigens comprising proteins, andthe second set of surface antigens comprises surface antigens with amolecular weight of less than 10,000 Da.

In yet another embodiment of any of the compositions provided, the firstset of surface antigens comprises surface antigens comprising proteins,and the second set of surface antigens comprises surface antigenscomprising peptides, proteins, oligosaccharides, polysaccharides and/orsmall molecules. In one embodiment, at least one surface antigen of thesecond set of surface antigens has a molecular weight of less than10,000 Da.

In still another embodiment of any of the compositions provided, thefirst set of surface antigens comprises surface antigens comprisingoligosaccharides, and the second set of surface antigens comprisessurface antigens with a molecular weight of less than 10,000 Da.

In a further embodiment of any of the compositions provided, the firstset of surface antigens comprises surface antigens comprisingoligosaccharides, and the second set of surface antigens comprisessurface antigens comprising peptides, proteins, oligosaccharides,polysaccharides and/or small molecules. In one embodiment, at least onesurface antigen of the second set of surface antigens has a molecularweight of less than 10,000 Da.

In yet a further embodiment of any of the compositions provided, thefirst set of surface antigens comprises surface antigens comprisingpolysaccharides, and the second set of surface antigens comprisessurface antigens with a molecular weight of less than 10,000 Da.

In still a further embodiment of any of the compositions provided, thefirst set of surface antigens comprises surface antigens comprisingpolysaccharides, and the second set of surface antigens comprisessurface antigens comprising peptides, proteins, oligosaccharides,polysaccharides and/or small molecules. In one embodiment, at least onesurface antigen of the second set of surface antigens has a molecularweight of less than 10,000 Da.

In another embodiment of any of the compositions provided, the first setof surface antigens comprises surface antigens comprising smallmolecules, and the second set of surface antigens comprises surfaceantigens with a molecular weight of less than 10,000 Da.

In still another embodiment of any of the compositions provided, thefirst set of surface antigens comprises surface antigens comprisingsmall molecules, and the second set of surface antigens comprisessurface antigens comprising peptides, proteins, oligosaccharides,polysaccharides and/or small molecules. In one embodiment, at least onesurface antigen of the second set of surface antigens has a molecularweight of less than 10,000 Da.

In one embodiment of any of the compositions provided, the compositionsfurther comprise one or more adjuvants. In one embodiment, the firstpopulation of synthetic nanocarriers and/or the second population ofsynthetic nanocarriers further comprise an adjuvant coupled to thesynthetic nanocarriers. In another embodiment, the first population ofsynthetic nanocarriers and/or the second population of syntheticnanocarriers further comprise an adjuvant coupled to the syntheticnanocarriers and the composition comprises one or more admixedadjuvants.

In one embodiment, each of the one or more adjuvants of any of thecompositions provided comprises a mineral salt, alum, alum combined withmonphosphoryl lipid (MPL) A of Enterobacteria, MPL® (AS04), AS15, asaponin, QS-21,Quil-A, ISCOMs, ISCOMATRIX™, MF59™, Montanide® ISA 51,Montanide® ISA 720, AS02, a liposome or liposomal formulation, AS01,AS15, synthesized or specifically prepared microparticles andmicrocarriers, bacteria-derived outer membrane vesicles of N. gonorrheaeor Chlamydia trachomatis, chitosan particles, a depot-forming agent,Pluronic® block co-polymers, specifically modified or prepared peptides,muramyl dipeptide, an aminoalkyl glucosaminide 4-phosphate, RC529, abacterial toxoid, a toxin fragment, an agonist of Toll-Like Receptors 2,3, 4, 5, 7, 8 or 9, an adenine derivative, immunostimulatory DNA,immunostimulatory RNA, an imidazoquinoline amine, an imidazopyridineamine, a 6,7-fused cycloalkylimidazopyridine amine, a 1,2-bridgedimidazoquinoline amine, imiquimod, resiquimod, an agonist for DC surfacemolecule CD40, a type I interferon, poly I:C, a bacteriallipopolysaccharide (LPS), VSV-G, HMGB-1, flagellin or portions orderivatives thereof, an immunostimulatory DNA molecule comprising CpG,proinflammatory stimuli released from necrotic cells, urate crystals, anactivated component of the complement cascade, an activated component ofimmune complexes, a complement receptor agonist, a cytokine, or acytokine receptor agonist. In one embodiment, the adjuvants aredifferent. In another embodiment, the adjuvant coupled to the firstpopulation of synthetic nanocarriers and/or the adjuvant coupled to thesecond population of synthetic nanocarriers comprises a TLR-2, -3, -4,-7, -8 or -9 agonist. In yet another embodiment, the adjuvant coupled tothe first population of synthetic nanocarriers and/or the adjuvantcoupled to the second population of synthetic nanocarriers comprises animmunostimulatory nucleic acid, imidazoquinoline, oxoadenine, MPL,imiquimod or resiquimod. In one embodiment, the admixed adjuvant is animmunostimulatory nucleic acid comprising CpG, AS01, AS02, AS04, AS15,QS-21, a saponin, alum or MPL.

In one embodiment of any of the compositions provided, the first andsecond populations of synthetic nanocarriers are present in an amounteffective to generate an immune response to the first set of surfaceantigens and the second set of surface antigens in a subject. In oneembodiment, the immune response is the generation of antibody titersspecific for the first set of surface antigens and the second set ofsurface antigens.

In another embodiment of any of the compositions provided, thecompositions comprise one or more additional populations of syntheticnanocarriers, wherein each additional population of syntheticnanocarriers comprises a set of surface antigens structurally differentfrom the other sets of surface antigens in the composition. In oneembodiment, at least one of the one or more additional populations ofsynthetic nanocarriers further comprise an adjuvant coupled thereto. Inanother embodiment, the adjuvant coupled to the at least one of the oneor more additional populations of synthetic nanocarriers is differentfrom the other adjuvants in the composition.

In yet another embodiment of any of the compositions provided, the firstset of surface antigens comprises a first set of monovalent oroligovalent surface antigens; and the second set of surface antigenscomprises a second set of monovalent or oligovalent surface antigens.

In still another embodiment of any of the compositions provided, eachset of surface antigens is a monovalent or oligovalent set of surfaceantigens.

In a further embodiment of any of the compositions provided, thepopulations of synthetic nanocarriers are present in an amount effectiveto generate an immune response to each set of surface antigens. In oneembodiment, the immune response is the generation of antibody titersspecific for each set of surface antigens.

In one embodiment of any of the compositions provided, the first and/orsecond population of synthetic nanocarriers further comprise a universalT cell antigen coupled thereto. In another embodiment, the universal Tcell antigen comprises a T helper cell antigen. In yet anotherembodiment, the T-helper cell antigen comprises a peptide obtained orderived from ovalbumin. In still another embodiment, the peptideobtained or derived from ovalbumin comprises the sequence as set forthin SEQ ID NO: 1. In a further embodiment, the universal T cell antigenis coupled by encapsulation.

In one embodiment of any of the compositions provided, thepharmaceutically acceptable excipient comprises a preservative, abuffer, saline, phosphate buffered saline, a colorant, or a stabilizer.

In another embodiment of any of the compositions provided, syntheticnanocarriers of each of the populations of synthetic nanocarrierscomprise lipid-based nanoparticles, polymeric nanoparticles, metallicnanoparticles, surfactant-based emulsions, dendrimers, buckyballs,nanowires, virus-like particles, peptide or protein-based particles,lipid-polymer nanoparticles, spheroidal nanoparticles, cuboidalnanoparticles, pyramidal nanoparticles, oblong nanoparticles,cylindrical nanoparticles, or toroidal nanoparticles. In one embodiment,each of the populations of synthetic nanocarriers comprise one or morepolymers. In another embodiment, the one or more polymers comprise apolyester. In still another embodiment, the one or more polymerscomprise or further comprise a polyester coupled to a hydrophilicpolymer. In yet another embodiment, the polyester comprises apoly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid),or polycaprolactone. In a further embodiment, the hydrophilic polymercomprises a polyether. In still a further embodiment, the polyethercomprises polyethylene glycol.

In another aspect, a method comprising administering any of thecompositions provided to a subject is provided. In one embodiment, thesubject has or is at risk of having an infection or infectious disease.In another embodiment, the subject has or is at risk of having cancer.In yet another embodiment, the subject has or is at risk of having anaddiction. In a further embodiment, the composition is administered byoral, subcutaneous, pulmonary, intranasal, intradermal or intramuscularadministration.

In yet another aspect, a method comprising preparing a first populationof synthetic nanocarriers that comprise a first set of surface antigens;preparing a second population of synthetic nanocarriers that comprise asecond set of surface antigens; and combining the first and secondpopulations of synthetic nanocarriers into a dosage form, wherein thefirst set of surface antigens and the second set of surface antigens arestructurally different is provided.

In a further aspect, a method comprising preparing a first population ofsynthetic nanocarriers that comprise a first set of surface antigens;preparing a second population of synthetic nanocarriers that comprise asecond set of surface antigens; and combining the first and secondpopulations of synthetic nanocarriers into a dosage form, wherein thefirst set of surface antigens and the second set of surface antigens areimmunologically different is provided. In one embodiment, the methodfurther comprises administering the dosage form to a subject. In anotherembodiment, the method further comprises determining whether or not animmune response to each set of surface antigens is generated. In oneembodiment, the immune response is the generation of antibody titersspecific for each set of surface antigens. In a further embodiment, themethod further comprises determining the amount effective to generatethe immune response to each set of surface antigens.

In still a further aspect, a process for producing a dosage form of acomposition, the process comprising the method steps as defined in anyof the methods provided herein is provided.

In one embodiment, any of the compositions provided is for use intherapy or prophylaxis. In another embodiment, any of the compositionsprovided is for use in any of the methods provided herein. In yetanother embodiment, any of the compositions provided is for use in amethod of treating or preventing infection or infectious disease. Instill another embodiment, any of the compositions provided is for use ina method of treating or preventing cancer. In a further embodiment, anyof the compositions provided is for use in a method of treating orpreventing an addiction.

In another embodiment, any of the methods comprise administration of anyof the compositions by oral, subcutaneous, pulmonary, intranasal,intradermal or intramuscular administration.

In yet another aspect, the use of any of the compositions provided forthe manufacture of a medicament for use in any of the methods providedis provided.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows anti-nicotine (dark gray bars) and anti-ovalbumin (lightgray bars) antibody titers in unimmunized mice and mice injected withNC-Nic and NC-OVA (5 animals/group; s.c., 100 μg of each NC perinjection, 2 times at 3-wk intervals).

FIG. 2 shows anti-nicotine, anti-ovalbumin, and anti-L2 peptide antibodytiters in unimmunized mice and mice injected with NC-Nic-OVA and NC-L2(5 animals/group; s.c., 100 μg of each NC per injection, 2 times at 3-wkintervals).

FIG. 3 shows anti-nicotine, anti-ovalbumin, anti-M2e peptide, andanti-L2 peptide antibody titers in unimmunized mice and mice injectedwith NC-Nic-OVA and NC-M2e-L2 (5 animals/group; s.c., 100 μg of each NCper injection, 2 times at 3-wk intervals).

FIG. 4 shows anti-M2e peptide and anti-L2 peptide antibody titers inunimmunized mice and mice injected with NC-M2e and NC-L2 (5animals/group; s.c., 100 μg of each NC per injection, 2 times at 3-wkintervals).

FIG. 5 shows anti-HA5 protein and anti-ovalbumin protein antibody titersin unimmunized mice and mice injected with NC-HA5 and NC-OVA (5animals/group; s.c., 100 μg of each NC per injection, 2 times at 3-wkintervals).

FIG. 6 shows anti-HA, anti-ovalbumin, anti-M2e peptide, and anti-L2peptide antibody titers in unimmunized mice and mice injected withNC-HA5, NC-OVA, and NC-M2e-L2 (5 animals/group; s.c., 100 μg of each NCper injection, 2 times at 3-wk intervals).

FIG. 7 shows antibody titers in mice immunized with a combination ofNC-M2e, NC-L2 peptide and NC-nicotine-ovalbumin.

FIG. 8 shows antibody titers in mice immunized with a combination ofNC-3′-nicotine and NC-1′-nicotine.

DETAILED DESCRIPTION OF THE INVENTION

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particularlyexemplified materials or process parameters as such may, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments of the inventiononly, and is not intended to be limiting of the use of alternativeterminology to describe the present invention.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entiretyfor all purposes.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural referents unless the contentclearly dictates otherwise. For example, reference to “a polymer”includes a mixture of two or more such molecules, reference to “asolvent” includes a mixture of two or more such solvents, reference to“an adhesive” includes mixtures of two or more such materials, and thelike.

Introduction

The inventors have unexpectedly and surprisingly discovered that theproblems and limitations noted above can be overcome by practicing theinvention disclosed herein. In particular, the inventors haveunexpectedly discovered that it is possible to provide inventivecompositions, and related methods, that address the problems andlimitations in the art by providing a composition comprising a dosageform comprising: a first population of synthetic nanocarriers thatcomprise a first set of surface antigens; a second population ofsynthetic nanocarriers that comprise a second set of surface antigens;and a pharmaceutically acceptable excipient; wherein the first set ofsurface antigens and the second set of surface antigens are structurallydifferent.

In another aspect, the invention provides a composition comprising: adosage form comprising: a first population of synthetic nanocarriersthat comprise a first set of surface antigens; a second population ofsynthetic nanocarriers that comprise a second set of surface antigens;and a pharmaceutically acceptable excipient; wherein the first set ofsurface antigens and the second set of surface antigens areimmunologically different.

In an aspect, the invention provides a composition comprising: a dosageform comprising: a first synthetic nanocarrier means for presenting afirst set of surface antigens; a second synthetic nanocarrier means forpresenting a second set of surface antigens; and a pharmaceuticallyacceptable excipient; wherein the first set of surface antigens and thesecond set of surface antigens are structurally different.

In an aspect, the invention provides a composition comprising: a dosageform comprising: a first synthetic nanocarrier means for presenting afirst set of surface antigens; a second synthetic nanocarrier means forpresenting a second set of surface antigens; and a pharmaceuticallyacceptable excipient; wherein the first set of surface antigens and thesecond set of surface antigens are immunologically different.

In another aspect, the invention provides a method comprising: preparinga first population of synthetic nanocarriers that comprise a first setof surface antigens; preparing a second population of syntheticnanocarriers that comprise a second set of surface antigens; andcombining the first and second populations of synthetic nanocarriersinto a dosage form; wherein the first set of surface antigens and thesecond set of surface antigens are structurally different.

In yet another aspect, the invention provides a method comprising:preparing a first population of synthetic nanocarriers that comprise afirst set of surface antigens; preparing a second population ofsynthetic nanocarriers that comprise a second set of surface antigens;and combining the first and second populations of synthetic nanocarriersinto a dosage form; wherein the first set of surface antigens and thesecond set of surface antigens are immunologically different.

It has been discovered that it is possible to generate a first andsecond population of synthetic nanocarriers that comprise a first andsecond set of surface antigens, respectively, which can be combinedtogether with a pharmaceutically acceptable excipient to create a dosageform. This dosage form can, in certain embodiments, be useful as amultivalent vaccine. The inventors have further noted certain advantagesin the creation of the inventive dosage forms, particularly with respectto conventional multivalent vaccines. These include, but are not limitedto, minimizing vaccine volumes which is a problem in conventionalmultivalent vaccines, and minimizing protein-protein interactionspresent in conventional protein carrier-hapten multivalent vaccines thatcan lead to non-specific binding and precipitation.

A further advantage of the present invention is that combining differentpopulations of synthetic nanocarriers that comprise sets of surfaceantigens allows different methods to be used to couple different sets ofsurface antigens to different populations of synthetic nanocarriers.This can be a significant advantage for embodiments wherein incompatiblecoupling methods are required to couple sets of surface antigens topopulations of synthetic nanocarriers. As an example, vaccines forStreptococcus pneumonia (U.S. Pat. No. 6,132,723 to Alberta ResearchCouncil and WO 2008/143709 to Wyeth) contain multiple antigens. Sincethe attachment chemistry conditions are not the same for all of thepolysaccharide antigens (WO 2008/143709) coupling methods that wouldattach all of the surface antigens to a single population of syntheticnanocariers in a single coupling environment would be undesirable.Practicing embodiments of the present invention wherein differentpopulations of synthetic nanocarriers are first coupled to certain setsof surface antigens and then combined could ameliorate the problem notedin the art.

Another example of multivalent vaccines that could benefit from thisembodiment of the invention comprise vaccines against N. meningitideswhich is polysaccharide-based and multivalent. Such embodiments may beaimed either at N. meningitidis groups A and C (bivalent) or groups A,C, W135 and Y (tetravalent).

The examples illustrate certain embodiments according to the invention,wherein peptides, polysaccharides, small molecules, etc., are conjugatedto a first population of synthetic nanocarriers and/or a secondpopulation of synthetic nanocarriers. These populations are thencombined to form a composition according to the invention.

The present invention will now be described in more detail.

Definition

“Abused substance” is any substance taken by a subject (e.g., a human)for purposes other than those for which it is indicated or in a manneror in quantities other than directed by a physician. The abusedsubstance, in some embodiments, is an addictive substance. In someembodiments, the abused substance for inclusion in a nanocarrier is thecomplete molecule, analog or a portion thereof. “Addictive substance” isa substance that causes obsession, compulsion, or physical dependence orpsychological dependence. In some embodiments, the addictive substancefor inclusion in a nanocarrier is the complete molecule, analog or aportion thereof.

“Adjuvant” means an agent that does not constitute a specific antigen,but boosts the strength and longevity of immune response to anadministered antigen (e.g., a concomitantly administered antigen). Suchadjuvants may include, but are not limited to stimulators of patternrecognition receptors, such as Toll-like receptors, RIG-1 and NOD-likereceptors (NLR), mineral salts, such as alum, alum combined withmonphosphoryl lipid (MPL) A of Enterobacteria, such as Escherichia coli,Salmonella minnesota, Salmonella typhimurium, or Shigella flexneri orspecifically with MPL® (AS04), MPL A of above-mentioned bacteriaseparately, saponins, such as QS-21,Quil-A, ISCOMs, ISCOMATRIX™,emulsions such as MF59™, Montanide® ISA 51 and ISA 720, AS02(QS21+squalene+MPL®) , liposomes and liposomal formulations such asAS01, AS15, synthesized or specifically prepared microparticles andmicrocarriers such as bacteria-derived outer membrane vesicles (OMV) ofN. gonorrheae, Chlamydia trachomatis and others, or chitosan particles,depot-forming agents, such as Pluronic® block co-polymers, specificallymodified or prepared peptides, such as muramyl dipeptide, aminoalkylglucosaminide 4-phosphates, such as RC529, or proteins, such asbacterial toxoids or toxin fragments.

In embodiments, adjuvants comprise agonists for pattern recognitionreceptors (PRR), including, but not limited to Toll-Like Receptors(TLRs), specifically TLRs 2, 3, 4, 5, 7, 8, 9 and/or combinationsthereof. In other embodiments, adjuvants comprise agonists for Toll-LikeReceptors 3, agonists for Toll-Like Receptors 7 and 8, or agonists forToll-Like Receptor 9; preferably the recited adjuvants compriseimidazoquinolines; such as R848; adenine derivatives, such as thosedisclosed in U.S. Pat. No. 6,329,381 (Sumitomo Pharmaceutical Company),US Published Patent Application 2010/0075995 to Biggadike et al., or WO2010/018132 to Campos et al.; immunostimulatory DNA; orimmunostimulatory RNA.

In specific embodiments, synthetic nanocarriers incorporate as adjuvantscompounds that are agonists for toll-like receptors (TLRs) 7 & 8 (“TLR7/8 agonists”). Of utility are the TLR 7/8 agonist compounds disclosedin U.S. Pat. No. 6,696,076 to Tomai et al., including but not limited toimidazoquinoline amines, imidazopyridine amines, 6,7-fusedcycloalkylimidazopyridine amines, and 1,2-bridged imidazoquinolineamines. Preferred adjuvants comprise imiquimod and resiquimod (alsoknown as R848). In specific embodiments, synthetic nanocarriersincorporate a ligand for toll-like receptor (TLR)-9, such as CpGs, whichinduce type I interferon production, and stimulate T and B cellactivation leading to increased antibody production and cytotoxic T cellresponses (Krieg et al., CpG motifs in bacterial DNA trigger direct Bcell activation. Nature. 1995. 374:546-549; Chu et al. CpGoligodeoxynucleotides act as adjuvants that switch on T helper 1 (Thi)immunity. J. Exp. Med. 1997. 186:1623-1631; Lipford et al.CpG-containing synthetic oligonucleotides promote B and cytotoxic T cellresponses to protein antigen: a new class of vaccine adjuvants. Eur. J.Immunol. 1997. 27:2340-2344; Roman et al. Immunostimulatory DNAsequences function as T helper-1-promoting adjuvants. Nat. Med. 1997.3:849-854; Davis et al. CpG DNA is a potent enhancer of specificimmunity in mice immunized with recombinant hepatitis B surface antigen.J. Immunol. 1998. 160:870-876; Lipford et al., Bacterial DNA as immunecell activator. Trends Microbiol. 1998. 6:496-500; U.S. Pat. No.6,207,646 to Krieg et al.; U.S. Pat. No. 7,223,398 to Tuck et al.; U.S.Pat. No. 7,250,403 to Van Nest et al.; or U.S. Pat. No. 7,566,703 toKrieg et al.).

In specific embodiments, an adjuvant may be an agonist for the DCsurface molecule CD40. In certain embodiments, to stimulate immunityrather than tolerance, a synthetic nanocarrier incorporates an adjuvantthat promotes DC maturation (needed for priming of naive T cells) andthe production of cytokines, such as type I interferons, which promoteantibody immune responses and anti-viral immunity. In embodiments,adjuvants also may comprise immunostimulatory RNA molecules, such as butnot limited to dsRNA, ssRNA, poly I:C or poly I:poly C12U (available asAmpligen®, both poly I:C and poly I:polyC12U being known as TLR3stimulants), and/or those disclosed in F. Heil et al., “Species-SpecificRecognition of Single-Stranded RNA via Toll-like Receptor 7 and 8”Science 303(5663), 1526-1529 (2004); J. Vollmer et al., “Immunemodulation by chemically modified ribonucleosides andoligoribonucleotides” WO 2008033432 A2; A. Forsbach et al.,“Immunostimulatory oligoribonucleotides containing specific sequencemotif(s) and targeting the Toll-like receptor 8 pathway” WO 2007062107A2; E. Uhlmann et al., “Modified oligcoribonucleotide analogs withenhanced immunostimulatory activity” U.S. Pat. Appl. Publ. US2006241076; G. Lipford et al., “Immunostimulatory viral RNAoligonucleotides and use for treating cancer and infections” WO2005097993 A2; G. Lipford et al., “Immunostimulatory G,U-containingoligoribonucleotides, compositions, and screening methods” WO 2003086280A2.

In some embodiments, an adjuvant may be a TLR-4 agonist, such asbacterial lipopolysacharide (LPS), VSV-G, and/or HMGB-1. In someembodiments, adjuvants may comprise TLR-5 agonists, such as flagellin,or portions or derivatives thereof, including but not limited to thosedisclosed in U.S. Pat. Nos. 6,130,082, 6,585,980, and 7,192,725.

In some embodiments, adjuvants may be proinflammatory stimuli releasedfrom necrotic cells (e.g., urate crystals). In some embodiments,adjuvants may be activated components of the complement cascade (e.g.,CD21, CD35, etc.). In some embodiments, adjuvants may be activatedcomponents of immune complexes. The adjuvants also include complementreceptor agonists, such as a molecule that binds to CD21 or CD35. Insome embodiments, the complement receptor agonist induces endogenouscomplement opsonization of the synthetic nanocarrier. In someembodiments, adjuvants are cytokines, which are small proteins orbiological factors (in the range of 5 kD-20 kD) that are released bycells and have specific effects on cell-cell interaction, communicationand behavior of other cells. In some embodiments, the cytokine receptoragonist is a small molecule, antibody, fusion protein, or aptamer.

In embodiments, at least a portion of the dose of adjuvant may becoupled to synthetic nanocarriers, preferably, all of the dose ofadjuvant is coupled to synthetic nanocarriers. In other embodiments, atleast a portion of the dose of the adjuvant is not coupled to thesynthetic nanocarriers. In embodiments, the dose of adjuvant comprisestwo or more types of adjuvants. For instance, and without limitation,adjuvants that act on different TLR receptors may be combined. As anexample, in an embodiment a TLR 7/8 agonist may be combined with a TLR 9agonist. In another embodiment, a TLR 7/8 agonist may be combined with aTLR 4 agonist. In yet another embodiment, a TLR 9 agonist may becombined with a TLR 3 agonist.

“Administering” or “administration” means providing a substance to asubject in a manner that is pharmacologically useful.

“Amount effective” is any amount of a composition that produces one ormore desired immune responses. This amount can be for in vitro or invivo purposes. For in vivo purposes, the amount can be one that a healthpractitioner would believe may have a clinical benefit for a subject inneed of an antibody response specific to one or more antigens. “Antibodyresponse” means any immune response that results in the production orstimulation of B cells and/or the production of antibodies. Inembodiments, therefore, an amount effective is one that a healthpractitioner would believe may generate an antibody response against thesurface antigen(s) of the inventive compositions provided herein.Effective amounts can be monitored by routine methods. An amount that iseffective to produce one or more desired immune responses can also be anamount of a composition provided herein that produces a desiredtherapeutic endpoint or a desired therapeutic result. Therefore, inother embodiments, the amount effective in one that a clinician wouldbelieve would provide a therapeutic benefit (including a prophylacticbenefit) to a subject provided herein. Such subjects include those thathave or are at risk of having cancer, an infection or infectiousdisease.

The antigen(s) of any of the inventive compositions provided herein canin embodiments be in an amount effective. In some embodiments, theamount effective is one that a health practitioner would believe maygenerate antibody titers against the sets of surface antigens of thecompositions provided herein. “Antibody titer” means the production of ameasurable level of antibodies. Preferably, the antibody response orgeneration of the antibody titer is in a human. In some embodiments, theantibodies are antibodies are of a certain isotype, such as IgG or asubclass thereof. Methods for measuring antibody titers are known in theart and include Enzyme-linked Immunosorbent Assay (ELISA). Methods formeasuring antibody response are also described in some detail in theExamples. Preferably, the antibody response or antibody titer isspecific to a set of surface antigens. In some embodiments where thesynthetic nanocarriers also comprise a universal antigen in addition toa set of surface antigens against which a specific immune response, suchas an antibody response or antibody titer, the immune response isspecific to the set of surface antigens but not to the universalantigen.

Amounts effective will depend, of course, on the particular subjectbeing treated; the severity of a condition, disease or disorder; theindividual patient parameters including age, physical condition, sizeand weight; the duration of the treatment; the nature of concurrenttherapy (if any); the specific route of administration and like factorswithin the knowledge and expertise of the health practitioner. Thesefactors are well known to those of ordinary skill in the art and can beaddressed with no more than routine experimentation. It is generallypreferred that a “maximum dose” be used, that is, the highest safe doseaccording to sound medical judgment. It will be understood by those ofordinary skill in the art, however, that a patient may insist upon alower dose or tolerable dose for medical reasons, psychological reasonsor for virtually any other reasons.

“Antigen” means a B cell antigen or T cell antigen. In embodiments,antigens are coupled to the synthetic nanocarriers. In otherembodiments, antigens are not coupled to the synthetic nanocarriers. Inembodiments antigens are coadministered with the synthetic nanocarriers.In other embodiments antigens are not coadministered with the syntheticnanocarriers. “Type(s) of antigens” means molecules that share the same,or substantially the same, antigenic characteristics.

“At least a portion of the dose” means at least some part of the dose,ranging up to including all of the dose.

An “at risk” subject is one in which a health practitioner believes hasa chance of having the disease or condition provided herein including,but not limited to, an infection, infectious disease, cancer or anaddiction.

“B cell antigen” means any antigen that is or recognized by and triggersan immune response in a B cell (e.g., an antigen that is specificallyrecognized by a B cell receptor on a B cell). In some embodiments, anantigen that is a T cell antigen is also a B cell antigen. In otherembodiments, the T cell antigen is not also a B cell antigen. B cellantigens include, but are not limited to proteins, peptides, smallmolecules, carbohydrates, oligosaccharides and polysaccharides. In someembodiments, the B cell antigen comprises a non-protein antigen (i.e.,not a protein or peptide antigen). In some embodiments, the B cellantigen comprises a carbohydrate, oligosaccharide or polysaccharideassociated with an infectious agent. In some embodiments, the B cellantigen comprises a glycoprotein or glycopeptide associated with aninfectious agent. The infectious agent can be a bacterium, virus,fungus, protozoan, or parasite. In some embodiments, the B cell antigencomprises a poorly immunogenic antigen. In some embodiments, the B cellantigen comprises an abused or addictive substance or a portion oranalog thereof. Addictive substances include, but are not limited to,nicotine, a narcotic, a cough suppressant, a tranquilizer, and asedative. In some embodiments, the B cell antigen comprises a toxin,such as a toxin from a chemical weapon or natural sources. The B cellantigen may also comprise a hazardous environmental agent. In someembodiments, the B cell antigen comprises a self antigen. In otherembodiments, the B cell antigen comprises an alloantigen, an allergen, acontact sensitizer, a degenerative disease antigen, a hapten, aninfectious disease antigen, a cancer antigen, an atopic disease antigen,an autoimmune disease antigen, an addictive substance, a xenoantigen, ora metabolic disease enzyme or enzymatic product thereof.

“Couple” or “Coupled” or “Couples” (and the like) means to chemicallyassociate one entity (for example a moiety) with another. In someembodiments, the coupling is covalent, meaning that the coupling occursin the context of the presence of a covalent bond between the twoentities. In non-covalent embodiments, the non-covalent coupling ismediated by non-covalent interactions including but not limited tocharge interactions, affinity interactions, metal coordination, physicaladsorption, host-guest interactions, hydrophobic interactions, TTstacking interactions, hydrogen bonding interactions, van der Waalsinteractions, magnetic interactions, electrostatic interactions,dipole-dipole interactions, and/or combinations thereof. In embodiments,encapsulation is a form of coupling.

“Derived” means adapted or modified from the original source. Forexample, as a non-limiting example, a peptide antigen derived from aninfectious strain may have several non-natural amino acid residuessubstituted for the natural amino acid residues found in the originalantigen found in the infectious strain. The adaptations or modificationsmay be for a variety of reasons, including but not limited to increasedspecificity, easier antigen processing, or improved safety.

In embodiments, a peptide or nucleic acid with a sequence with only 50%identity to a natural peptide or nucleic acid, preferably a naturalconsensus peptide or nucleic acid, would be said to be derived from thenatural peptide or nucleic acid. In other embodiments, the material issubstantially modified. Substantially modified material means a materialthat is modified such that the modification significantly affects thechemical or immunological properties of the material in question.Derived peptides and nucleic acids can also include those with asequence with greater than 50% identity to a natural peptide or nucleicacid sequence if said derived peptides and nucleic acids have alteredchemical or immunological properties as compared to the natural peptideor nucleic acid. These chemical or immunological properties comprisehydrophilicity, stability, affinity, and ability to couple with acarrier such as a synthetic nanocarrier.

“Dosage form” means a pharmacologically and/or immunologically activematerial, such as a vaccine, in a medium, carrier, vehicle, or devicesuitable for administration to a subject.

“Encapsulate” or “encapsulated” means to enclose within a syntheticnanocarrier, preferably enclose completely within a syntheticnanocarrier. Most or all of a substance that is encapsulated is notexposed to the local environment external to the synthetic nanocarrier.Encapsulation is distinct from adsorption, which places most or all of asubstance on a surface of a synthetic nanocarrier, and leaves thesubstance exposed to the local environment external to the syntheticnanocarrier.

“Immunologically different” refers to a difference between certainsurface antigens that can be noted if a sera generated by immunizationgenerates a distinct antibody response spectrum for each of the surfaceantigens. Surface antigen specific antibodies will only recognize aspecific set of surface antigens, and will bind in distinguishablebinding patterns to other sets of surface antigens. For example, ifimmunized with a set of surface antigens A, the antiserum generated willbind to the set of surface antigens A, but not to the set of surfaceantigens B. If two or more surface antigens are combined on a singlesynthetic nanocarrier, a panning assay can be designed which willdistinguish the binding patterns of the sera relative to the two sets ofsurface antigens. In embodiments, a first set of surface antigens and asecond set of surface antigens are immunologically different. In otherembodiments, a first set of surface antigens, a second set of surfaceantigens, and a third set of surface antigens all are immunologicallydifferent.

An “infection” or “infectious disease” is any condition or diseasecaused by a microorganism, pathogen or other agent, such as a bacterium,fungus, prion or virus. The surface antigens if the inventivecompositions provided herein can be obtained or derived from anyinfectious agent, such as those that can cause the infections orinfectious diseases provided herein.

“Infectious genus” means a genus that comprises organisms capable ofinfecting a subject. In embodiments, surface antigens may be obtained orderived from a first infectious genus, or obtained or derived from asecond infectious genus. In embodiments, the first infectious genus andthe second infectious genus are the same. In other embodiments, thefirst infectious genus and the second infectious genus are different.

“Infectious species” means a species that comprises organisms capable ofinfecting a subject. In embodiments, surface antigens may be obtained orderived from a first infectious species, or obtained or derived from asecond infectious species. In embodiments, the first infectious speciesand the second infectious species are of the same genus. In otherembodiments, the first infectious species and the second infectiousspecies are also the same. In some embodiments, the first infectiousspecies and the second infectious species are different but are of thesame genus. In other embodiments, the different infectious species areof different genera.

“Infectious strain” means a strain that comprises organisms capable ofinfecting a subject. In embodiments, surface antigens may be obtained orderived from a first infectious strain, or obtained or derived from asecond infectious strain. In embodiments, the first infectious strainand the second infectious strain are of the same species. In otherembodiments, the first infectious strain and the second infectiousstrain are also the same. In still other embodiments, they are of thesame species but are of a different strain. In some embodiments, thefirst infectious strain and the second infectious strain are ofdifferent species but of the same genus.

“Isolated nucleic acid” means a nucleic acid that is separated from itsnative environment and present in sufficient quantity to permit itsidentification or use. An isolated nucleic acid may be one that is (i)amplified in vitro by, for example, polymerase chain reaction (PCR);(ii) recombinantly produced by cloning; (iii) purified, as by cleavageand gel separation; or (iv) synthesized by, for example, chemicalsynthesis. An isolated nucleic acid is one which is readily manipulableby recombinant DNA techniques well known in the art. Thus, a nucleotidesequence contained in a vector in which 5′ and 3′ restriction sites areknown or for which polymerase chain reaction (PCR) primer sequences havebeen disclosed is considered isolated but a nucleic acid sequenceexisting in its native state in its natural host is not. An isolatednucleic acid may be substantially purified, but need not be. Forexample, a nucleic acid that is isolated within a cloning or expressionvector is not pure in that it may comprise only a tiny percentage of thematerial in the cell in which it resides. Such a nucleic acid isisolated, however, as the term is used herein because it is readilymanipulable by standard techniques known to those of ordinary skill inthe art. Any of the nucleic acids provided herein may be isolated. Insome embodiments, the antigens in the compositions provided herein arepresent in the form of an isolated nucleic acid, such as an isolatednucleic acid that encodes an antigenic peptide, polypeptide or protein.

“Isolated peptide, polypeptide or protein” means the peptide,polypeptide or protein is separated from its native environment andpresent in sufficient quantity to permit its identification or use. Thismeans, for example, the peptide, polypeptide or protein may be (i)selectively produced by expression cloning or (ii) purified as bychromatography or electrophoresis. Isolated peptides, polypeptides orproteins may be, but need not be, substantially pure. Because anisolated peptide, polypeptide or protein may be admixed with apharmaceutically acceptable carrier in a pharmaceutical preparation, thepeptide, polypeptide or protein may comprise only a small percentage byweight of the preparation. The peptide, polypeptide or protein isnonetheless isolated in that it has been separated from the substanceswith which it may be associated in living systems, i.e., isolated fromother peptides, polypeptides or proteins. Any of the peptides,polypeptides or proteins provided herein may be isolated. In someembodiments, the antigens in the compositions provided herein arepeptides, polypeptides or proteins.

“Maximum dimension of a synthetic nanocarrier” means the largestdimension of a nanocarrier measured along any axis of the syntheticnanocarrier. “Minimum dimension of a synthetic nanocarrier” means thesmallest dimension of a synthetic nanocarrier measured along any axis ofthe synthetic nanocarrier. For example, for a spheroidal syntheticnanocarrier, the maximum and minimum dimension of a syntheticnanocarrier would be substantially identical, and would be the size ofits diameter. Similarly, for a cuboidal synthetic nanocarrier, theminimum dimension of a synthetic nanocarrier would be the smallest ofits height, width or length, while the maximum dimension of a syntheticnanocarrier would be the largest of its height, width or length. In anembodiment, a minimum dimension of at least 75%, preferably at least80%, more preferably at least 90%, of the synthetic nanocarriers in asample, based on the total number of synthetic nanocarriers in thesample, is greater than 100 nm. In a embodiment, a maximum dimension ofat least 75%, preferably at least 80%, more preferably at least 90%, ofthe synthetic nanocarriers in a sample, based on the total number ofsynthetic nanocarriers in the sample, is equal to or less than 5 μm.Preferably, a minimum dimension of at least 75%, preferably at least80%, more preferably at least 90%, of the synthetic nanocarriers in asample, based on the total number of synthetic nanocarriers in thesample, is equal to or greater than 110 nm, more preferably equal to orgreater than 120 nm, more preferably equal to or greater than 130 nm,and more preferably still equal to or greater than 150 nm. Aspectsratios of the maximum and minimum dimensions of inventive syntheticnanocarriers may vary depending on the embodiment. For instance, aspectratios of the maximum to minimum dimensions of the syntheticnanocarriers may vary from 1:1 to 1,000,000:1, preferably from 1:1 to100,000:1, more preferably from 1:1 to 1000:1, still preferably from 1:1to 100:1, and yet more preferably from 1:1 to 10:1. Preferably, amaximum dimension of at least 75%, preferably at least 80%, morepreferably at least 90%, of the synthetic nanocarriers in a sample,based on the total number of synthetic nanocarriers in the sample isequal to or less than 3 μm, more preferably equal to or less than 2 μm,more preferably equal to or less than 1 μm, more preferably equal to orless than 800 nm, more preferably equal to or less than 600 nm, and morepreferably still equal to or less than 500 nm. In preferred embodiments,a maximum dimension of at least 75%, preferably at least 80%, morepreferably at least 90%, of the synthetic nanocarriers in a sample,based on the total number of synthetic nanocarriers in the sample, isequal to or greater than 100 nm, more preferably equal to or greaterthan 120 nm, more preferably equal to or greater than 130 nm, morepreferably equal to or greater than 140 nm, and more preferably stillequal to or greater than 150 nm. Measurement of synthetic nanocarriersizes is obtained by suspending the synthetic nanocarriers in a liquid(usually aqueous) media and using dynamic light scattering (e.g. using aBrookhaven ZetaPALS instrument).

“Molecular weight less than 10,000” means a molecular weight calculatedbased on the molecular structure of a molecule of less than 10,000.

“Obtained” means taken from a source without substantial modification.Substantial modification is modification that significantly affects thechemical or immunological properties of the material in question. Forexample, as a non-limiting example, a peptide or nucleic acid with asequence with greater than 90%, preferably greater than 95%, preferablygreater than 97%, preferably greater than 98%, preferably greater than99%, preferably 100%, identity to a natural peptide or nucleotidesequence, preferably a natural consensus peptide or nucleotide sequence,and chemical and/or immunological properties that are not significantlydifferent from the natural peptide or nucleic acid would be said to beobtained from the natural peptide or nucleotide sequence. These chemicalor immunological properties comprise hydrophilicity, stability,affinity, and ability to couple with a carrier such as a syntheticnanocarrier. In embodiments, the obtained material has been taken fromthe original source and has not been adapted or modified. For example,in embodiments, antigens obtained from a source may comprise theoriginal amino acid residue sequence found in that source. In otherembodiments, for example, antigens obtained from a source may comprisethe original molecular structure found in that source.

“Oligosaccharide(s)” means a saccharide polymer containing a smallnumber (typically two to twenty) of saccharide units linked byglycosidic bonds. At a high number of saccharide units, oligosaccharidesmay comprise polysaccharides.

“Peptide(s)” means compounds comprising amino acid residues joinedtogether primarily by peptide bonds between the carboxyl and aminogroups of adjacent amino acid residues, and possessing 100 or less aminoacid residues. Certain of the peptide bonds in peptide may be replacedby other bond types, for various purposes, such as stabilization orcoupling.

“Pharmaceutically acceptable excipient (or carrier)” means apharmacologically inactive material used together with the recitedsynthetic nanocarriers to formulate the inventive compositions.Pharmacologically inactive materials can be added to an inventive dosageform to further facilitate administration of the composition.Pharmaceutically acceptable excipients comprise a variety of materialsknown in the art, including but not limited to saccharides (such asglucose, lactose, and the like), preservatives such as antimicrobialagents, reconstitution aids, colorants, saline (such as phosphatebuffered saline), and buffers. Examples, without limitation, ofpharmaceutically acceptable excipients include calcium carbonate,calcium phosphate, various diluents, various sugars and types of starch,cellulose derivatives, gelatin, vegetable oils, polyethylene glycols,preservatives, various pharmaceutical carriers, sterile saline,lyophilization stabilizers, and the like. The compositions may be madeusing conventional pharmaceutical manufacturing and compoundingtechniques to arrive at useful dosage forms. In an embodiment, inventivesynthetic nanocarriers are suspended in sterile saline solution forinjection together with a preservative.

“Polysaccharide(s)” means a saccharide polymer made of many saccharideunits linked by glycosidic bonds. At a low number of saccharide units,polysaccharides may comprise oligosaccharides.

“Population” means a defined group of synthetic nanocarriers that shareone or more common physical or chemical characteristics. Common physicalor chemical characteristics may comprise having a common set of surfaceantigens, common coupled adjuvant(s), common materials making up thebulk nanocarrier, a common shape, a common particle size, and the like.Multiple populations of synthetic nanocarriers may be identified, forexample a first population, a second population, a third population, afourth population, and the like. In an embodiment, three or morepopulations of synthetic nanocarriers may be present, preferably whereineach population of synthetic nanocarriers comprises a set of surfaceantigens; and wherein each set of surface antigens is structurally orimmunologically different from one another.

“Protein(s)” means compounds, typically having a molecular weightgreater than 1000 daltons, comprising amino acid residues joinedtogether primarily by peptide bonds between the carboxyl and aminogroups of adjacent amino acid residues. Proteins may also compriseadditional bonding structures such as secondary structures, tertiarystructures, and the like. Certain of the peptide bonds in proteins maybe replaced by other bond types, for various purposes, such asstabilization or coupling.

“Set of monovalent surface antigens” means a set of surface antigens inwhich the surface antigens are not different, preferably not differenteither structurally and/or immunologically. In embodiments, the set ofmonovalent surface antigens is composed of multiple copies of one typeof surface antigen that is not different structurally or immunologically(i.e., multiple copies of the same antigen). The multiple copies of thissame antigen can be in some embodiments strung together, such as thatillustrated in US Publication 2003/0223938. A set of monovalent surfaceantigens that is composed of multiple copies of one type of surfaceantigen that is not different structurally or immunologically is not aset of oligovalent (or multivalent) surface antigens.

“Set of oligovalent (or multivalent) surface antigens” means a set ofsurface antigens in which there are a limited number, that is greaterthan one, of different types of surface antigens, preferably wherein thedifference comprises structural difference and/or immunologicaldifference. In preferred embodiments, the limited number of surfaceantigens in the set comprise 2 to 15 types of surface antigens,preferably 2 to 10 types of surface antigens, more preferably 2 to 8types of surface antigens, more preferably 2 to 7 types of surfaceantigens, more preferably 2 to 6 types of surface antigens, morepreferably 2 to 5 types of surface antigens, more preferably 2 to 4types of surface antigens, more preferably 2 to 3 types of surfaceantigens, and even more preferably 2 types of surface antigens. In otherembodiments, the set of oligovalent (or multivalent surface antigens)comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19 or 20 or more types of surface antigens.

“Set of surface antigens” means a group of surface antigens that areidentified, preferably identified through measurement and/or prediction,based on their properties, preferably their structural and/or theirimmunological properties. A set of surface antigens may be identified,in part or in whole, based on prediction using the chemical syntheticmethods used to synthesize, along with the chemical methods used tocouple, the set of surface antigens and/or the population of syntheticnanocarriers of which the set of surface antigens are comprised.Multiple sets of surface antigens can be identified; e.g. a first set, asecond set, a third set, and so on.

“Structurally different” or “structural difference” means presentingdifferent molecular structures for interaction with a B cell receptor.In embodiments, this difference can be expressed by comparing theprevalence and types of presented antigens in a set of surface antigensto the prevalence and types of presented antigens in a different set ofsurface antigens. If the prevalence and/or types of presented antigensare different between the sets, then the sets of surface antigens can besaid to be structurally different. In embodiments, the difference in theprevalence and/or types of presented antigens can be ascertained bycomparison of the chemical synthetic strategies and formulationstrategies used to generate the surface antigens and/or couple thesurface antigens to a surface of the synthetic nanocarriers. Forexample, in an embodiments, if a set of surface antigens was generatedusing a particular chemical compound or compounds, and another set ofsurface antigens was generated using a different chemical compound orcompounds, then the two sets of surface antigens could be ascertained tobe different. In a different embodiment, if surface antigens weregenerated using three chemical compounds to form a set of three surfaceantigens, and other surface antigens were generated using two chemicalcompounds to form a set of two surface antigens, then the two sets ofsurface antigens could be ascertained to be different. In yet anotherembodiment, if non-differing chemical synthetic strategies andformulation strategies (including using non-different amounts ofmaterials—within experimental error—in the strategies) are used togenerate two sets of surface antigens and (as appropriate) couple thetwo sets of surface antigens to surfaces of synthetic nanocarriers, andthe two sets of surface antigens possessed the same conformation andorientation, then the two sets of surface antigens likely would not bestructurally different. In embodiments, the structural differencebetween a first set of surface antigens and a second set of surfaceantigens comprises non-differing sets of molecules that are presented inorientations that differ between the first and second sets of surfaceantigens. In embodiments, the structural difference between a first setof surface antigens and a second set of surface antigens comprisesnon-differing sets of molecules that are presented in conformations thatdiffer between the first and second sets of surface antigens. Inembodiments, the structural difference between a first set of surfaceantigens and a second set of surface antigens comprises sets ofmolecules whose molecular structure is different between the first andsecond sets of surface antigens.

“Subject” means animals, including warm blooded mammals such as humansand primates; avians; domestic household or farm animals such as cats,dogs, sheep, goats, cattle, horses and pigs; laboratory animals such asmice, rats and guinea pigs; fish; reptiles; zoo and wild animals; andthe like.

“Surface antigen(s)” means an antigen found on or around a surface of asynthetic nanocarrier. In preferable embodiments, surface antigenscomprise B cell antigens. In embodiments, surface antigens are coupledto a surface of the synthetic nanocarriers.

“Synthetic nanocarrier(s)” means a discrete object that is not found innature, and that possesses at least one dimension that is less than orequal to 5 microns in size. Albumin nanoparticles are generally includedas synthetic nanocarriers, however in certain embodiments the syntheticnanocarriers do not comprise albumin nanoparticles. In embodiments, theinventive synthetic nanocarriers do not comprise chitosan.

A synthetic nanocarrier can be, but is not limited to, one or aplurality of lipid-based nanoparticles (e.g. liposomes) (also referredto herein as lipid nanoparticles, i.e., nanoparticles where the majorityof the material that makes up their structure are lipids), polymericnanoparticles, metallic nanoparticles, surfactant-based emulsions,dendrimers, buckyballs, nanowires, virus-like particles (i.e., particlesthat are primarily made up of viral structural proteins but that are notinfectious or have low infectivity), peptide or protein-based particles(also referred to herein as protein particles, i.e., particles where themajority of the material that makes up their structure are peptides orproteins) (such as albumin nanoparticles) and/or nanoparticles that aredeveloped using a combination of nanomaterials such as lipid-polymernanoparticles. Synthetic nanocarriers may be a variety of differentshapes, including but not limited to spheroidal, cuboidal, pyramidal,oblong, cylindrical, toroidal, and the like. Synthetic nanocarriersaccording to the invention comprise one or more surfaces. Exemplarysynthetic nanocarriers that can be adapted for use in the practice ofthe present invention comprise: (1) the biodegradable nanoparticlesdisclosed in U.S. Pat. No. 5,543,158 to Gref et al., (2) the polymericnanoparticles of Published US Patent Application 20060002852 to Saltzmanet al., (3) the lithographically constructed nanoparticles of PublishedUS Patent Application 20090028910 to DeSimone et al., (4) the disclosureof WO 2009/051837 to von Andrian et al., (5) the nanoparticles disclosedin Published US Patent Application 2008/0145441 to Penades et al., (6)the protein nanoparticles disclosed in Published US Patent Application20090226525 to de los Rios et al., (7) the virus-like particlesdisclosed in published US Patent Application 20060222652 to Sebbel etal., (8) the nucleic acid coupled virus-like particles disclosed inpublished US Patent Application 20060251677 to Bachmann et al., (9) thevirus-like particles disclosed in WO2010047839A1 or WO2009106999A2, or(10) the nanoprecipitated nanoparticles disclosed in P. Paolicelli etal., “Surface-modified PLGA-based Nanoparticles that can EfficientlyAssociate and Deliver Virus-like Particles” Nanomedicine. 5(6):843-853(2010). In embodiments, synthetic nanocarriers may possess an aspectratio greater than 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5, 1:7, or greaterthan 1:10.

Synthetic nanocarriers according to the invention that have a minimumdimension of equal to or less than about 100 nm, preferably equal to orless than 100 nm, do not comprise a surface with hydroxyl groups thatactivate complement or alternatively comprise a surface that consistsessentially of moieties that are not hydroxyl groups that activatecomplement. In a preferred embodiment, synthetic nanocarriers accordingto the invention that have a minimum dimension of equal to or less thanabout 100 nm, preferably equal to or less than 100 nm, do not comprise asurface that substantially activates complement or alternativelycomprise a surface that consists essentially of moieties that do notsubstantially activate complement. In a more preferred embodiment,synthetic nanocarriers according to the invention that have a minimumdimension of equal to or less than about 100 nm, preferably equal to orless than 100 nm, do not comprise a surface that activates complement oralternatively comprise a surface that consists essentially of moietiesthat do not activate complement. In embodiments, synthetic nanocarriersexclude virus-like particles. In embodiments, when syntheticnanocarriers comprise virus-like particles, the virus-like particlescomprise non-natural adjuvant (meaning that the VLPs comprise anadjuvant other than naturally occurring RNA generated during theproduction of the VLPs). In embodiments, synthetic nanocarriers maypossess an aspect ratio greater than 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5,1:7, or greater than 1:10.

“T cell antigen” means any antigen that is recognized by and triggers animmune response in a T cell (e.g., an antigen that is specificallyrecognized by a T cell receptor on a T cell or an NKT cell viapresentation of the antigen or portion thereof bound to a Class I orClass II major histocompatability complex molecule (MHC), or bound to aCD1 complex. In some embodiments, an antigen that is a T cell antigen isalso a B cell antigen. In other embodiments, the T cell antigen is notalso a B cell antigen. T cell antigens generally are proteins orpeptides. T cell antigens may be an antigen that stimulates a CD8+ Tcell response, a CD4+ T cell response, or both. The nanocarriers,therefore, in some embodiments can effectively stimulate both types ofresponses.

In some embodiments the T cell antigen is a ‘universal’ T cell antigen,or T cell memory antigen, (i.e., one to which a subject has apre-existing memory and that can be used to boost T cell help to anunrelated antigen, for example an unrelated B cell antigen). Universal Tcell antigens include tetanus toxoid, as well as one or more peptidesderived from tetanus toxoid, Epstein-Barr virus, or influenza virus.Universal T cell antigens also include a components of influenza virus,such as hemagglutinin, neuraminidase, or nuclear protein, or one or morepeptides derived therefrom. In some embodiments, the universal T cellantigen is not one that is presented in a complex with a MHC molecule.In some embodiments, the universal T cell antigen is not complexed witha MHC molecule for presentation to a T helper cell. Accordingly, in someembodiments, the universal T cell antigen is not a T helper cellantigen. However, in other embodiments, the universal T cell antigen isa T helper cell antigen.

In embodiments, a T-helper cell antigen may comprise one or morepeptides obtained or derived from tetanus toxoid, Epstein-Barr virus,influenza virus, respiratory syncytial virus, measles virus, mumpsvirus, rubella virus, cytomegalovirus, adenovirus, diphtheria toxoid, ora PADRE peptide (known from the work of Sette et al. U.S. Pat. No.7,202,351). In other embodiments, a T-helper cell antigen may compriseovalbumin or a peptide obtained or derived therefrom. Preferably, theovalbumin comprises the amino acid sequence as set forth in AccessionNo. AAB59956, NP_(—)990483.1, AAA48998, or CAA2371. In otherembodiments, the peptide obtained or derived from ovalbumin comprisesthe following amino acid sequence:H-Ile-Ser-Gln-Ala-Val-His-Ala-Ala-His-Ala-Glu-Ile-Asn-Glu-Ala-Gly-Arg-OH(SEQ ID NO: 1). In other embodiments, a T-helper cell antigen maycomprise one or more lipids, or glycolipids, including but not limitedto: α-galactosylceramide (α-GalCer), α-linked glycosphingolipids (fromSphingomonas spp.), galactosyl diacylglycerols (from Borreliaburgdorferi), lypophosphoglycan (from Leishmania donovani), andphosphatidylinositol tetramannoside (PIM4) (from Mycobacterium leprae).For additional lipids and/or glycolipids useful as T-helper cellantigen, see V. Cerundolo et al., “Harnessing invariant NKT cells invaccination strategies.” Nature Rev Immun, 9:28-38 (2009).

In embodiments, CD4+ T-cell antigens may be derivatives of a CD4+ T-cellantigen that is obtained from a source, such as a natural source. Insuch embodiments, CD4+ T-cell antigen sequences, such as those peptidesthat bind to MHC II, may have at least 70%, 80%, 90%, or 95% identity tothe antigen obtained from the source. In embodiments, the T cellantigen, preferably a universal T cell antigen or T-helper cell antigen,may be coupled to, or uncoupled from, a synthetic nanocarrier. In someembodiments, the universal T cell antigen or T-helper cell antigen isencapsulated in the synthetic nanocarriers of the inventivecompositions.

“Types of surface antigens,” “surface antigen types,” etc. means adefined group of surface antigens that share one or more common chemicaland/or immunological characteristics.

“Vaccine” means a composition of matter that improves the immuneresponse to a particular pathogen or disease. A vaccine typicallycontains factors that stimulate a subject's immune system to recognize aspecific antigen as foreign and eliminate it from the subject's body. Avaccine also establishes an immunologic ‘memory’ so the antigen will bequickly recognized and responded to if a person is re-challenged.Vaccines can be prophylactic (for example to prevent future infection byany pathogen), or therapeutic (for example a vaccine against a tumorspecific antigen for the treatment of cancer). In embodiments, a vaccinemay comprise dosage forms according to the invention.

Nanocarrier Populations and Sets of Surface Antigens

In embodiments, populations of synthetic nanocarriers share commonphysical or chemical characteristics. In embodiments, such commonphysical or chemical characteristics may comprise a common set ofsurface antigens, common coupled adjuvant(s), common materials making upthe bulk nanocarrier, a common shape, a common particle size, commonsurface charge, and the like. Types of adjuvants, materials, shapes andparticle sizes are discussed throughout the present application.

In embodiments, a population may share a set of common surface antigens.These common surface antigens may be grouped together based on commonphysical or chemical characteristics, such as, but not limited to,structural or immunological properties. In embodiments, the commoncharacteristics may comprise common orientation or conformation, or setsof molecules sharing a common molecular structure, or all of theforegoing. In embodiments, common surface antigens may comprise thosehaving a molecular weight less than 10,000. In other embodiments, commonsurface antigens may comprise being peptides, proteins,oligosaccharides, polysaccharides, or small molecules. In still otherembodiments, common surface antigens may comprise those having amolecular weight less than 10,000 and that comprise being peptides,proteins, oligosaccharides, polysaccharides, or small molecules. Inother embodiments, the common surface antigens may be grouped togetherbased on the infectious organisms that they were obtained or derivedfrom; and would be categorized as sharing a common genus, species,and/or strain. In embodiments wherein the surface antigens have amolecular weight less than 10,000, common surface antigens may begrouped together based on classes of molecules such as chemical warfareagents, environmental toxins, addictive or abused substances, andphysiologically endogenous molecules including but not limited tohormones, lipids and neurotransmitters. In embodiments, sets of commonsurface antigens may be defined by the strength of their ability toinduce an antibody response in vivo. For example one set of surfaceantigens may have the ability to ability to induce high affinityantibody production in vivo, while another set of common surfaceantigens may induce low affinity antibody production in vivo.

In embodiments, a set of surface antigens (e.g., a first and/or secondset of surface antigens) can comprise antigens obtained or derived froman infectious agent. In some embodiments, the infectious agent is abacterium, fungus, virus, protozoan, or parasite. In other embodiments,the virus is a pox virus, smallpox virus, ebola virus, marburg virus,measles virus (in embodiments, the antigen can be obtained or derivedfrom hemagglutinin protein, hemagglutinin noose epitope, hemagglutiningamino acids 106-114 and/or 519-550, etc.), dengue fever virus, influenzavirus, influenza A virus (in embodiments, the antigen can be obtained orderived from HA protein, M2e protein, etc.), influenza H5N1 virus,influenza H1N1 virus, infectious salmon anemia virus, parainfluenzavirus, respiratory syncytial virus, rubeola virus, humanimmunodeficiency virus, human papillomavirus, varicella-zoster virus,herpes simplex virus, cytomegalovirus, Epstein-Barr virus, JC virus,rhabdovirus, rotavirus, rhinovirus, adenovirus, papillomavirus (inembodiments, the antigen can be obtained or derived from L1 or L2protein), parvovirus, picornavirus, poliovirus, virus that causes mumps,virus that causes rabies, reovirus, rubella virus, togavirus,orthomyxovirus, retrovirus, hepadnavirus, coxsackievirus, equineencephalitis virus, tick-borne encephalitis, Japanese encephalitisvirus, yellow fever virus, Rift Valley fever virus, hepatitis A virus,hepatitis B virus, hepatitis C virus, hepatitis D virus, or hepatitis Evirus.

In embodiments, a set of surface antigens (e.g., a first and/or secondset of surface antigens) comprises or is obtained or derived from avirus of a family of viruses shown below in Table 1. In anotherembodiment, a set of surface antigens (e.g., a first and/or second setof surface antigens) comprises or is obtained or derived from a virus ofa species provided in Table 1. In still another embodiment, a set ofsurface antigens (e.g., a first and/or second set of surface antigens)comprises or is obtained or derived from an antigen provided in Table 1.

TABLE 1 Viral Infectious Agents Family Exemplary Species ExemplaryAntigens Adenoviridae adenovirus VI, VII, E1A, E3-19K, 52KPicornaviridae coxsackievirus, , VP1 hepatitis A virus Surface antigenpoliovirus, 3A protein, capsid protein Rhinovirus (e.g., nucleocapsid,surface projection, type 16) and transmembrane proteins HerpesviridaeHerpes simplex Capsid proteins (e.g., UL6, (type 1 and type 2) UL18,UL35, UL38, and UL19) Varicella-zoster Early antigen virus Epstein-barrvirus, Early antigen, capsid antigen Human Pp65, gB, p52 cytomegalovirusHuman herpesvirus, Latent nuclear antigen-1 (e.g., type 8)Hepadnaviridae Hepatitis B virus surface antigen Flaviviridae HepatitisC virus, NS3, Envelop protein (e.g., E2 yellow fever virus, domain)dengue virus, West Nile virus Retroviridae HIV gp120, p24, andlipopeptides Gag (17-35), Gag (253-284), Nef (66-97), Nef (116-145), andPol (325-355); see Roberts et al., J. Immunol. Methods, 365(1-2): 27-37,2011 Orthomyxoviridae Influenza virus neuraminidase, surface antigen,Measles virus, Nucleocapsid protein, matrix Mumps virus protein,phosphoprotein, fusion Parainfluenza virus protein, hemagglutinin,hemagglutinin-neuraminidase, glycoprotein, Paramyxoviridae Respiratorysyncytial virus Human metapneumovirus Papillomaviridae Human E6, E7,capsid antigen papillomavirus (e.g., type 16 and 18) RhabdoviridaeRabies virus Envelope lipoprotein Togaviridae Rubella virus Capsidprotein Paroviridae Human bocarivus, Capsid protein, non-structuralParvovirus B19 protein (NS)

In other embodiments, a set of surface antigens (e.g., a first and/orsecond set of surface antigens) can comprise antigens obtained orderived from bacterial organisms such as Borrelia species, Bacillusanthracis, Borrelia burgdorferi, Bordetella pertussis, Bordetellaparapertussis, Camphylobacter jejuni, Chlamydia species, Chlamydialpsittaci, Chlamydial trachomatis, Clostridium species, Clostridiumtetani, Clostridium botulinum, Clostridium perfringens, Corynebacteriumdiphtheriae, Coxiella species, an Enterococcus species, Erlichiaspecies, Escherichia coli, Francisella tularensis, Haemophilus species,Haemophilus influenzae, Haemophilus parainfluenzae, Lactobacillusspecies, a Legionella species, Legionella pneumophila, Leptospirosisinterrogans, Listeria species, Listeria monocytogenes, Mycobacteriumspecies, Mycobacterium tuberculosis, Mycobacterium leprae, Mycoplasmaspecies, Mycoplasma pneumoniae, Neisseria species, Neisseriameningitidis, Neisseria gonorrhoeae, Pneumococcus species (e.g., type6A, 6B, 3, 4, 14, 19F, etc.), Pseudomonas species, Pseudomonasaeruginosa, Salmonella species, Salmonella typhi, Salmonella enterica,Rickettsia species, Rickettsia ricketsii, Rickettsia typhi, Shigellaspecies, Staphylococcus species, Staphylococcus aureus, Streptococcusspecies, Streptococccus pnuemoniae, Streptococcus pyrogenes,Streptococcus mutans, Treponema species, Treponema pallidum, a Vibriospecies, Vibrio cholerae, Yersinia pestis, and the like.

In embodiments, a set of surface antigens (e.g., a first and/or secondset of surface antigens) comprises or is obtained or derived from abacteria of a genera of bacteria shown below in Table 2. In anotherembodiment, a set of surface antigens (e.g., a first and/or second setof surface antigens) comprises or is obtained or derived from abacterial species provided in Table 2. In still another embodiment, aset of surface antigens (e.g., a first and/or second set of surfaceantigens) comprises or is obtained or derived from an antigen providedin Table 2.

TABLE 2 Bacterial Infectious Agents Pathogenic Bacterial GeneraExemplary Species Exemplary Antigens Bordetella Bordetella pertussispertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (PRN),and fimbriae (FIM 2/3) Borrelia Borrelia burgdorferi VlsE; DbpA and OspABrucella Brucella abortus Hia, PrpA, MltA, L7/L12, D15, 0187, VirJ, Mdh,AfuA Brucella canis L7/L12 Brucella melitensis Out membrane proteinssuch as Omp28 Brucella suis Campylobacter Campylobacter jejuni; LPS, an100-kD antigen Chlamydia and Chlamydia pneumoniae See Richard et al., J.Infectious Chlamydophila Chlamydia trachomatis Diseases. 181: S521(2000) Chlamydophila psittaci Clostridium Clostridium botulinum antigentypes A, B, C, D, and E Clostridium difficile FliC, FliD, and Cwp84Clostridium perfringens alpha-toxin, theta-toxin, fructose 1,6-biphosphate-aldolase (FBA), glyceraldehydes-3-phosphate dehydrogenase(GPD), pyruvate: ferredoxin oxidoreductase (PFOR), elongation factor-G(EF-G), and a hypothetical protein (HP) Clostridium tetani T toxinCorynebacterium Corynebacterium diphtheriae Toxoid antigen EnterococcusEnterococcus faecalis capsular polysaccharides Enterococcus faeciumEscherichia Escherichia coli See Moriel et al., PNAS 107(20): 9072- 9077(2010) Francisella Francisella tularensis See Havlasova et al.,Proteomics 2(7): 857-867, 2002 Haemophilus Haemophilus influenzaecapsular polysaccharides, Protein D, Helicobacter Helicobacter pyloriSee Bumann et al., Proteomics 4(10): 2843-2843, 2004 LegionellaLegionella pneumophila Mip Leptospira Leptospira interrogans See Brownet al., Infect Immu 59(5): 1772-1777, 1991 Listeria Listeriamonocytogenes nucleoprotein (NP) Mycobacterium* Mycobacterium lepraeMycobacterium tuberculosis RD1, PE35, PPE68, EsxA, EsxB, RD9, and EsxVMycobacterium ulcerans Mycoplasma Mycoplasma pneumoniae Hsp70 NeisseriaNeisseria gonorrhoeae Neisseria meningitidis See Litt et al., J.Infectious Disease 190(8): 1488-1497, 2004 Pseudomonas Pseudomonasaeruginosa Lipopolysaccharides Rickettsia Rickettsia rickettsii Surfaceantigen Salmonella Salmonella typhi Salmonella typhimurium ShigellaShigella sonnei Staphylococcus Staphylococcus aureus See Vytvtska etal., Proteomics 2(5): 580-590, 2002; Etz et al., PNAS 99(10): 6573-6578;2002 Staphylococcus epidermidis Staphylococcus saprophyticusStreptococcus Streptococcus agalactiae Streptococcus pneumoniae Sp1,Sp2, Sp3 Streptococcus pyogenes Lei et al., J. Infectious Disease189(1): 79-89, 2004 Treponema Treponema pallidum GlycerophosphodiesterPhosphodiesterase Vibrio Vibrio cholerae Outer membrane proteins such asOmpK Yersinia Yersinia pestis Chaperone-usher protein, capsular protein(F1), and V protein

In still other embodiments, a set of surface antigens (e.g., a firstand/or second set of surface antigens) can comprise antigens obtained orderived from antigens of fungal, protozoan, and/or parasitic organismssuch as Aspergillus species, Candida species, Candida albicans, Candidatropicalis, Cryptococcus species, Cryptococcus neoformans, Entamoebahistolytica, Histoplasma capsulatum, Leishmania species, Nocardiaasteroides, Plasmodium falciparum, Toxoplasma gondii, Trichomonasvaginalis, Toxoplasma species, Trypanosoma brucei, Schistosoma mansoni,and the like.

In still other embodiments, a set of surface antigens (e.g., a firstand/or second set of surface antigens) can comprise antigens obtained orderived from a toxin, such as O-Alkyl (<C10, incl. cycloalkyl) alkyl(Me, Et, n-Pr or i-Pr)-phosphonofluoridates (e.g. Sarin: O-Isopropylmethylphosphonofluoridate, Soman: O-Pinacolylmethylphosphonofluoridate), O-Alkyl (<C10, incl. cycloalkyl) N,N-dialkyl(Me, Et, n-Pr or i-Pr) phosphoramidocyanidates (e.g. Tabun: O-EthylN,N-dimethylphosphoramidocyanidate), O-Alkyl (H or <C10, incl.cycloalkyl) S-2-dialkyl (Me, Et, n-Pr or i-Pr)-aminoethyl alkyl (Me, Et,n-Pr or i-Pr) phosphonothiolates and corresponding alkylated orprotonated salts (e.g. VX: O-Ethyl S-2-diisopropylaminoethylmethylphosphonothiolate), Sulfur mustards:2-Chloroethylchloromethylsulfide, Mustard gas:Bis(2-chloroethyl)sulfide, Bis(2-chloroethylthio)methane, Sesquimustard:1,2-Bis(2-chloroethylthio)ethane, 1,3-Bis(2-chloroethylthio)-n-propane,1,4-Bis(2-chloroethylthio)-n-butane,1,5-Bis(2-chloroethylthio)-n-pentane, Bis(2-chloroethylthiomethyl)ether,O-Mustard: Bis(2-chloroethylthioethyl)ether, Lewisites: Lewisite 1:2-Chlorovinyldichloroarsine, Lewisite 2: Bis(2-chlorovinyl)chloroarsine,Lewisite 3: Tris(2-chlorovinyl)arsine, Nitrogen mustards: HN1:Bis(2-chloroethyl)ethylamine, HN2: Bis(2-chloroethyl)methylamine, HN3:Tris(2-chloroethyl)amine, Saxitoxin, Ricin, Amiton: O,O-DiethylS-(2-(diethylamino)ethyl)phosphorothiolate and corresponding alkylatedor protonated salts, PFIB:1,1,3,3,3-Pentafluoro-2-(trifluoromethyl)-1-propene, 3-Quinuclidinylbenzilate (BZ), Phosgene: Carbonyl dichloride, Cyanogen chloride,Hydrogen cyanide and Chloropicrin: Trichloronitromethane.

In other embodiments, a set of surface antigens (e.g., a first and/orsecond set of surface antigens) comprises or is obtained or derived froma fungus of a genera of fungi shown below in Table 3. In anotherembodiment, a set of surface antigens (e.g., a first and/or second setof surface antigens) comprises or is obtained or derived from a fungalspecies provided in Table 3. In still another embodiment, a set ofsurface antigens (e.g., a first and/or second set of surface antigens)comprises or is obtained or derived from an antigen provided in Table 3.

TABLE 3 Fungal Infectious Agents Genera Exemplary Species ExemplaryAntigens Candida C. albicans Surface antigens, see also Thomas et al.,Proteomics 6(22): 6033-6041, 2006 Aspergillus Aspergillus fumigatusStevens et al., Medical and Aspergillus flavus. Mycology 49 (Suppl. 1):S170-S176, 2011 Cryptococcus Cryptococcus Capsular glycoproteins,neoformans, Cryptococcus laurentii and Cryptococcus albidus,Cryptococcus gattii Histoplasma Histoplasma Yps3P, Hsp60 capsulatumPneumocystis Pneumocystis Major surface proteins jirovecii (Msg) such asMsgC1, MsgC3, MsgC8, and MsgC9 Stachybotrys Stachybotrys SchS34,chartarum

In still further embodiments, a set of surface antigens (e.g., a firstand/or second set of surface antigens) can comprise antigens obtained orderived from an abused or addictive substance. In some embodiments, theabused or addictive substance is a drug, such as an illegal drug, anover-the-counter drug or a prescription drug. In other embodiments, theabused or addictive substance has mood-altering effects, and, therefore,includes inhalants and solvents. In other embodiments, the abused oraddictive substance is one that has no mood-altering effects orintoxication properties, and, therefore, includes anabolic steroids.Abused or addictive substances include, but are not limited to,cannabinoids (e.g., hashish, marijuana), depressants (e.g., barbituates,benodiazepines, flunitrazepam (Rohypnol), GHB, methaqualone(quaaludes)), dissociative anesthetics (e.g., ketamine, PCP),hallucinogens (e.g, LSD, mescaline, psilocybin), opioids and morphinederivatives (e.g., codeine, fentanyl, heroin, morphine, opium),stimulants (amphetamine, cocaine, Ecstacy (MDMA), methamphetamine,methylphenidate (Ritalin), nicotine), anabolic steriods, and inhalants.In embodiments, the antigen comprises a cocaine analog, such asnorcocaine. In other embodiments, the antigen comprises cotinine.

In embodiments of the present invention, different populations ofsynthetic nanocarriers that each comprise a set of surface antigens maybe combined. The difference between the populations is based on thedifferences between the sets of surface antigens.

In certain embodiments, these differences can comprise differences inphysical or chemical characteristics, such as, but not limited to,structural or immunological properties. In embodiments, the differencesmay comprise differences in surface antigen orientation or conformation,or differences in molecular structure between sets of surface antigens.In still other embodiments, the difference in surface antigens may bebased on the infectious organisms that they were obtained or derivedfrom; and would be categorized as being from a different genus, species,and/or strain. In embodiments wherein the surface antigens have amolecular weight less than 10,000, surface antigens may be differentbased on chemical classes such as chemical warfare agents, addictive orabused substances, and endogenous molecules including but not limited tohormones, lipids and neurotransmitters.

In embodiments, the differences may comprise differences in surfaceantigen orientation or conformation. For instance, different points ofattachment of a surface antigen to a synthetic nanocarrier would giverise to different presentations of that surface antigen. These differentpresentations may produce antibodies that recognize different epitopesof the surface antigen. Surface antigens may be presented with differentconformations, and may be synthesized or modified to achieve thoseconformations. For example, peptide or protein truncations may beperformed in which results in modified conformational changes in thepeptide or protein antigen of interest. Alternatively amino acids orchemical linkers may be added in order to add length or stabilize aspecific orientation that alters peptide or protein antigen exposure.Similarly antigens such those as having a molecular weight less than10,000, or oligosaccharides, or polysaccharides may be altered byaddition of a chemical linker, or chemical modification.

In other embodiments, differences between populations of syntheticnanocarriers may be based on differences between sets of surfaceantigens based on different molecular structures and/or prevalence ofthe antigens. In some embodiments, the difference may comprise adifference in the prevalence of one or more of the types of surfaceantigens between the sets.

In embodiments wherein a population comprises a monovalent set ofsurface antigens, the molecular structure, preferably the antigen type,of its set of surface antigens may be different from the molecularstructure of a set of monovalent surface antigens of another populationor other populations. In certain embodiments, wherein a populationcomprises a monovalent set of surface antigens, the prevalence ofsurface antigens of which its set of surface antigens is comprised maybe different from the prevalence of surface antigens of which a set ofmonovalent surface antigens of another population or other populationsis comprised.

In embodiments wherein a population of synthetic nanocarriers comprisesa set of oligovalent (or multivalent) surface antigens, various antigentypes at different prevalences may be combined within the set to formcombinations of such surface antigen types. Accordingly, in embodimentswherein at least one population of synthetic nanocarriers comprises aset of oligovalent (or multivalent) surface antigens, the molecularstructure of the set of oligovalent (or multivalent) surface antigens(which can be expressed as a function of both the molecular structure ofeach type of antigen, along with their prevalence within the set) can bedifferent from the molecular structure of the set of surface antigens ofanother population or other populations. In embodiments, this may bebecause another population comprises a monovalent set of surfaceantigens (wherein the sets of surface antigens would be different bydefinition) or because another population comprises a set of oligovalent(or multivalent) surface antigens wherein the molecular structures ofthe two sets of surface antigens (expressed as a molecular structure ofeach type of antigen and/or a prevalence of each antigen type within theset) are different.

For example the sets of surface antigens can be comprised of a set ofenantiomers such as (R) and (S) nicotine. The enantiomers can be presentin equal amounts on the same or different nanocarriers and can bepresent in unequal amounts on the same or different populations ofsynthetic nanocarriers. In another embodiment, a set of surface antigensmay comprise two structurally different but related molecules such ascotinine and nicotine, either optically pure or racemic. The cotinineand nicotine can be present in equal amounts on the same or differentpopulations of synthetic nanocarriers and can be present in unequalamounts on the same or different populations of synthetic nanocarriers.In addition, sets of surface antigens can be comprised of antigens froma single organism comprised of several serotypes such as the capsularantigenic polysaccharides from Streptococcus Pneumoniae serotypes 4, 6B,9V,14, 18C, 19F, and 23F. The various antigens can be present in equalamounts on the same or different populations of synthetic nanocarriersand can be present in unequal amounts on the same or differentpopulations of synthetic nanocarriers. In embodiments, sets of surfaceantigens can comprise a family of different antigens from a singleorganism such as the capsid proteins L1 and L2 of the humanpapillomavirus. The sets of surface antigens can be present in equalamounts on the same or different populations of synthetic nanocarriersand can be present in unequal amounts on the same or differentpopulations of synthetic nanocarriers. In embodiments, sets of surfaceantigens can comprise several small molecules of diverse structures suchas the war gases VX, sarin and soman. The different compounds can bepresent in equal amounts on the same or different populations ofsynthetic nanocarriers and can be present in unequal amounts on the sameor different populations of synthetic nanocarriers. For instance, in anembodiment, one set of surface antigens may comprise 50% of VX and 50%sarin, while another set of surface antigens may comprise 80% of VX and20% sarin, where the percent of surface antigens may be a weight percentor mole percent, and based on the total weight or total number of molesof surface antigens.

In embodiments, differences in sets of surface antigens may compriseproviding a population of synthetic nanocarriers that comprises a set ofsurface antigens from a type or types such as having a molecular weightless than 10,000 and/or being peptides, proteins, oligosaccharides,polysaccharides, or small molecules; and then providing anotherpopulation of synthetic nanoparticles comprising a different set ofsurface antigens from a type or types such as having a molecular weightless than 10,000 and/or or being peptides, proteins, oligosaccharides,polysaccharides, or small molecules.

In embodiments, when a first set of surface antigens comprises surfaceantigens having a molecular weight less than 10,000, a second set ofsurface antigens comprises peptides, proteins, oligosaccharides,polysaccharides or small molecules (provided the sets are structurallyor immunologically different). In embodiments, when a first set ofsurface antigens comprises surface antigens comprising peptides, asecond set of surface antigens comprises surface antigens comprisingthose having a molecular weight less than 10,000 and/or comprisingproteins, oligosaccharides, polysaccharides or small molecules. Inembodiments, when a first set of surface antigens comprises surfaceantigens comprising proteins, a second set of surface antigens comprisessurface antigens comprising those having a molecular weight less than10,000 and/or comprising peptides, oligosaccharides, polysaccharides orsmall molecules. In embodiments, when a first set of surface antigenscomprises surface antigens comprising oligosaccharides, a second set ofsurface antigens comprises surface antigens comprising those having amolecular weight less than 10,000 and/or comprising peptides, proteins,polysaccharides or small molecules. In embodiments, when a first set ofsurface antigens comprises surface antigens comprising polysaccharides,a second set of surface antigens comprises surface antigens comprisingthose having a molecular weight less than 10,000 and/or comprisingpeptides, proteins, oligosaccharides or small molecules. In embodiments,when a first set of surface antigens comprises surface antigenscomprising small molecules, a second set of surface antigens comprisessurface antigens comprising those having a molecular weight less than10,000 and/or comprising peptides, proteins, oligosaccharides orpolysaccharides (provided the sets are structurally or immunologicallydifferent).

In embodiments, when a second set of surface antigens comprises surfaceantigens having a molecular weight less than 10,000, a first set ofsurface antigens comprises peptides, proteins, oligosaccharides,polysaccharides or small molecules (provided the sets are structurallyor immunologically different). In embodiments, when a second set ofsurface antigens comprises surface antigens comprising peptides, a firstset of surface antigens comprises surface antigens comprising thosehaving a molecular weight less than 10,000 and/or comprising proteins,oligosaccharides, polysaccharides or small molecules. In embodiments,when a second set of surface antigens comprises surface antigenscomprising proteins, a first set of surface antigens comprises surfaceantigens comprising those having a molecular weight less than 10,000and/or comprising peptides, oligosaccharides, polysaccharides or smallmolecules. In embodiments, when a second set of surface antigenscomprises surface antigens comprising oligosaccharides, a first set ofsurface antigens comprises surface antigens comprising those having amolecular weight less than 10,000 and/or comprising peptides, proteins,polysaccharides or small molecules. In embodiments, when a second set ofsurface antigens comprises surface antigens comprising polysaccharides,a first set of surface antigens comprises surface antigens comprisingthose having a molecular weight less than 10,000 and/or comprisingpeptides, proteins, oligosaccharides or small molecules. In embodiments,when a second set of surface antigens comprises surface antigenscomprising small molecules, a first set of surface antigens comprisessurface antigens comprising those having a molecular weight less than10,000 and/or comprising peptides, proteins, oligosaccharides orpolysaccharides (provided the sets are structurally or immunologicallydifferent).

Other differences between populations of synthetic nanocarriers may bebased on differences between sets of surface antigens based on thesource of the antigens. For instance, in an embodiment, such differencesmay be based on differences of the infectious genera, species and/orstrains from which the surface antigens were obtained or derived. Forexample, one population of synthetic nanocarriers may comprise a set ofsurface antigens obtained or derived from a bacterial source, such as E.Coli, mycobacterium tuberculosis, clostridium tetani or bacillusanthracis while another population may comprise a set of surfaceantigens obtained or derived from a viral source, such as influenzavirus, hepatitis B virus, hepatitis C virus, and human herpesvirus. Inembodiments, one population of synthetic nanocarriers may comprise a setof surface antigens obtained or derived from a bacterial source, such asthose noted above, while another population may comprise a set ofsurface antigens obtained or derived from fungi such as candidaalbicans, or pneumocystis jiroveci. In other embodiments, one populationof synthetic nanocarriers may comprise a set of surface antigensobtained or derived from a viral source, while another population maycomprise a set of surface antigens obtained or derived from parasitessuch as plasmodium falciparum. Other combinations and sub-combinationsalong the lines of the illustrations above are contemplated to be withinthe scope of the present invention.

In other embodiments, the various sets of surfaces antigens may beobtained or derived from infectious genera, species or strains that arenot different. In embodiments, those differences may arise fromselection of different antigens within the infectious genus, species orstrain. For example, one set of surface antigens may be obtained orderived from one viral coat protein, while another set of surfaceantigens may be obtained or derived from different epitopes from thesame or another viral coat protein from the same virus. In anembodiment, different sets of surface antigens may be obtained orderived from one viral protein, for example, the cytomegalovirus (CMV)capsid protein, or other CMV proteins, which may comprise severaldistinct epitopes. Likewise, different sets of surface antigens may beobtained or derived from different epitopes of diphtheria or tetanustoxin.

In other embodiments, differences between populations of syntheticnanocarriers may be based on differences between sets of surfaceantigens based on different chemical classes of the antigens. Forinstance, in the case of molecules having a molecular weight less than10,000 such differences may be based on differences of the chemicalscaffold, or the overall molecular structure, or the activity exhibitedby such molecules. For instance, sets of different surface antigens maybe obtained or derived from sets of surface antigens of differingstructure but with similar activities like opiods such as morphine andheroin. In other embodiments, different sets of surface antigens may becomprised of molecules with similar structures but with differingactivities exemplified by enantiomers such as (R) and (S) Ritalin, or(R) and (S) nicotine. In embodiments, difference between sets of surfaceantigens may comprise compounds and their metabolites such asterfenadine and fexofenadine or astemazole and norastemazole.Differences between sets of surface antigens may also be based on theun-relatedness of the structure of compounds such as nicotine andmethamphetamine.

In other embodiments, differences between populations of syntheticnanocarriers may be based on differences between sets of surfaceantigens based on immunological differences between the sets of surfaceantigens. In embodiments, sets of surface antigens may be defined bytheir ability to induce an immune response in vivo. For example, one setof surface antigens may have the ability to induce high levels of highaffinity antibody production to an antigen of interest in vivo, while asecond set of surface antigens may not induce high levels of highaffinity antibody production in vivo to that antigen. As another examplea first set of surface antigens may have the ability to generateantibody titers specific to the antigens of the first set of surfaceantigens, while a second set of surface antigens may have the abilitygenerate antibody titers specific to the antigens of the second set ofsurface antigens. In embodiments, the second set of surface antigensgenerate antibody titers specific to the antigens of the second set ofsurface antigens but not to the antigens of the first set.

Inventive Compositions and Related Methods

Synthetic nanocarriers may be prepared using a wide variety of methodsknown in the art. For example, synthetic nanocarriers can be formed bymethods as nanoprecipitation, flow focusing using fluidic channels,spray drying, single and double emulsion solvent evaporation, solventextraction, phase separation, milling, microemulsion procedures,microfabrication, nanofabrication, sacrificial layers, simple andcomplex coacervation, and other methods well known to those of ordinaryskill in the art. Alternatively or additionally, aqueous and organicsolvent syntheses for monodisperse semiconductor, conductive, magnetic,organic, and other nanomaterials have been described (Pellegrino et al.,2005, Small, 1:48; Murray et al., 2000, Ann. Rev. Mat. Sci., 30:545; andTrindade et al., 2001, Chem. Mat., 13:3843). Additional methods havebeen described in the literature (see, e.g., Doubrow, Ed.,“Microcapsules and Nanoparticles in Medicine and Pharmacy,” CRC Press,Boca Raton, 1992; Mathiowitz et al., 1987, J. Control. Release, 5:13;Mathiowitz et al., 1987, Reactive Polymers, 6:275; and Mathiowitz etal., 1988, J. Appl. Polymer Sci., 35:755, and also U.S. Pat. Nos.5,578,325 and 6,007,845; P. Paolicelli et al., “Surface-modifiedPLGA-based Nanoparticles that can Efficiently Associate and DeliverVirus-like Particles” Nanomedicine. 5(6):843-853 (2010)).

In embodiments, the present invention comprises synthetic nanocarriermeans for presenting sets of surface antigens, preferably sets ofmonovalent or oligovalent surface antigens. A particular inventiveembodiment comprises a first synthetic nanocarrier means for presentinga first set of surface antigens, preferably a first set of monovalent oroligovalent surface antigens; and a second synthetic nanocarrier meansfor presenting a second set of surface antigens; preferably a second setof monovalent or oligovalent surface antigens. Such syntheticnanocarrier means for presenting surface antigens are disclosedthroughout the present disclosure, and encompass the embodimentsdisclosed herein.

Various materials may be encapsulated into synthetic nanocarriers asdesirable using a variety of methods including but not limited to C.Astete et al., “Synthesis and characterization of PLGA nanoparticles” J.Biomater. Sci. Polymer Edn, Vol. 17, No. 3, pp. 247-289 (2006); K.Avgoustakis “Pegylated Poly(Lactide) and Poly(Lactide-Co-Glycolide)Nanoparticles: Preparation, Properties and Possible Applications in DrugDelivery” Current Drug Delivery 1:321-333 (2004); C. Reis et al.,“Nanoencapsulation I. Methods for preparation of drug-loaded polymericnanoparticles” Nanomedicine 2:8-21 (2006); P. Paolicelli et al.,“Surface-modified PLGA-based Nanoparticles that can EfficientlyAssociate and Deliver Virus-like Particles” Nanomedicine. 5(6):843-853(2010). Other methods suitable for encapsulating materials, such asoligonucleotides, into synthetic nanocarriers may be used, includingwithout limitation methods disclosed in U.S. Pat. No. 6,632,671 to UngerOct. 14, 2003.

In certain embodiments, synthetic nanocarriers are prepared by ananoprecipitation process or spray drying. Conditions used in preparingsynthetic nanocarriers may be altered to yield particles of a desiredsize or property (e.g., hydrophobicity, hydrophilicity, externalmorphology, “stickiness,” shape, etc.). The method of preparing thesynthetic nanocarriers and the conditions (e.g., solvent, temperature,concentration, air flow rate, etc.) used may depend on the materials tobe coupled to the synthetic nanocarriers and/or the composition of thepolymer matrix.

If particles prepared by any of the above methods have a size rangeoutside of the desired range, particles can be sized, for example, usinga sieve, differential centrifugation or settling.

Elements of the inventive synthetic nanocarriers such as moieties ofwhich an immunofeature surface is comprised, targeting moieties,polymeric matrices, antigens, adjuvants, and the like may be coupled tothe synthetic nanocarrier, e.g., by one or more covalent bonds, or maybe coupled by means of one or more linkers. Additional methods offunctionalizing synthetic nanocarriers may be adapted from Published USPatent Application 2006/0002852 to Saltzman et al., Published US PatentApplication 2009/0028910 to DeSimone et al., or Published InternationalPatent Application WO/2008/127532 A1 to Murthy et al.

Alternatively or additionally, synthetic nanocarriers can be coupled tomoieties of which an immunofeature surface is comprised, targetingmoieties, adjuvants, antigens and/or other elements directly orindirectly via non-covalent interactions. In non-covalent embodiments,the non-covalent coupling is mediated by non-covalent interactionsincluding but not limited to charge interactions, affinity interactions,metal coordination, physical adsorption, host-guest interactions,hydrophobic interactions, TT stacking interactions, hydrogen bondinginteractions, van der Waals interactions, magnetic interactions,electrostatic interactions, dipole-dipole interactions, and/orcombinations thereof. Such couplings may be arranged to be on anexternal surface or an internal surface of an inventive syntheticnanocarrier. In embodiments, encapsulation and/or absorption is a formof coupling.

A wide variety of synthetic nanocarriers can be used according to theinvention. In some embodiments, synthetic nanocarriers are spheres orspheroids. In some embodiments, synthetic nanocarriers are flat orplate-shaped. In some embodiments, synthetic nanocarriers are cubes orcuboidal. In some embodiments, synthetic nanocarriers are ovals orellipses. In some embodiments, synthetic nanocarriers are cylinders,cones, or pyramids.

In some embodiments, it is desirable to use a population of syntheticnanocarriers that is relatively uniform in terms of size, shape, and/orcomposition so that each synthetic nanocarrier has similar properties.For example, at least 80%, at least 90%, or at least 95% of thesynthetic nanocarriers, based on the total number of syntheticnanocarriers, may have a minimum dimension or maximum dimension thatfalls within 5%, 10%, or 20% of the average diameter or averagedimension of the synthetic nanocarriers. In some embodiments, apopulation of synthetic nanocarriers may be heterogeneous with respectto size, shape, and/or composition.

Synthetic nanocarriers can be solid or hollow and can comprise one ormore layers. In some embodiments, each layer has a unique compositionand unique properties relative to the other layer(s). To give but oneexample, synthetic nanocarriers may have a core/shell structure, whereinthe core is one layer (e.g. a polymeric core) and the shell is a secondlayer (e.g. a lipid bilayer or monolayer). Synthetic nanocarriers maycomprise a plurality of different layers.

In some embodiments, synthetic nanocarriers may optionally comprise oneor more lipids. In some embodiments, a synthetic nanocarrier maycomprise a liposome. In some embodiments, a synthetic nanocarrier maycomprise a lipid bilayer. In some embodiments, a synthetic nanocarriermay comprise a lipid monolayer. In some embodiments, a syntheticnanocarrier may comprise a micelle. In some embodiments, a syntheticnanocarrier may comprise a core comprising a polymeric matrix surroundedby a lipid layer (e.g., lipid bilayer, lipid monolayer, etc.). In someembodiments, a synthetic nanocarrier may comprise a non-polymeric core(e.g., metal particle, quantum dot, ceramic particle, bone particle,viral particle, proteins, nucleic acids, carbohydrates, etc.) surroundedby a lipid layer (e.g., lipid bilayer, lipid monolayer, etc.).

In some embodiments, synthetic nanocarriers can comprise one or morepolymers. In some embodiments, such a polymer can be surrounded by acoating layer (e.g., liposome, lipid monolayer, micelle, etc.). In someembodiments, various elements of the synthetic nanocarriers can becoupled with the polymer.

In some embodiments, an immunofeature surface, targeting moiety,antigens, adjuvants, and/or oligonucleotide can be covalently associatedwith a polymeric matrix. In some embodiments, covalent association ismediated by a linker. In some embodiments, an immunofeature surface,targeting moiety, antigens, adjuvants, and/or oligonucleotide can benoncovalently associated with a polymeric matrix. For example, in someembodiments, an immunofeature surface, targeting moiety, antigens,adjuvants, and/or oligonucleotide can be encapsulated within, surroundedby, and/or dispersed throughout a polymeric matrix. Alternatively oradditionally, an immunofeature surface, targeting moiety, antigens,adjuvants, and/or nucleotide can be associated with a polymeric matrixby hydrophobic interactions, charge interactions, van der Waals forces,etc.

A wide variety of polymers and methods for forming polymeric matricestherefrom are known conventionally. In general, a polymeric matrixcomprises one or more polymers. Polymers may be natural or unnatural(synthetic) polymers. Polymers may be homopolymers or copolymerscomprising two or more monomers. In terms of sequence, copolymers may berandom, block, or comprise a combination of random and block sequences.Typically, polymers in accordance with the present invention are organicpolymers.

Examples of polymers suitable for use in the present invention include,but are not limited to polyethylenes, polycarbonates (e.g.poly(1,3-dioxan-2one)), polyanhydrides (e.g. poly(sebacic anhydride)),polypropylfumerates, polyamides (e.g. polycaprolactam), polyacetals,polyethers, polyesters (e.g., polylactide, polyglycolide,polylactide-co-glycolide, polycaprolactone, polyhydroxyacids (e.g.poly(β-hydroxyalkanoate))), poly(orthoesters), polycyanoacrylates,polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates,polymethacrylates, polyureas, polystyrenes, polyamines, polylysine,polylysine-PEG copolymers, poly(ethyleneimine), poly(ethylene imine)-PEGcopolymers, and polyphosphazines.

In some embodiments, polymers in accordance with the present inventioninclude polymers which have been approved for use in humans by the U.S.Food and Drug Administration (FDA) under 21 C.F.R. §177.2600, includingbut not limited to polyesters (e.g., polylactic acid,poly(lactic-co-glycolic acid), polycaprolactone, polyvalerolactone,poly(1,3-dioxan-2one)); polyanhydrides (e.g., poly(sebacic anhydride));polyethers (e.g., polyethylene glycol); polyurethanes;polymethacrylates; polyacrylates; and polycyanoacrylates.

In some embodiments, polymers can be hydrophilic. For example, polymersmay comprise anionic groups (e.g., phosphate group, sulphate group,carboxylate group); cationic groups (e.g., quaternary amine group); orpolar groups (e.g., hydroxyl group, thiol group, amine group). In someembodiments, a synthetic nanocarrier comprising a hydrophilic polymericmatrix generates a hydrophilic environment within the syntheticnanocarrier. In some embodiments, polymers can be hydrophobic. In someembodiments, a synthetic nanocarrier comprising a hydrophobic polymericmatrix generates a hydrophobic environment within the syntheticnanocarrier. Selection of the hydrophilicity or hydrophobicity of thepolymer may have an impact on the nature of materials that areincorporated (e.g. coupled) within the synthetic nanocarrier.

In some embodiments, polymers may be modified with one or more moietiesand/or functional groups. A variety of moieties or functional groups canbe used in accordance with the present invention. In some embodiments,polymers may be modified with polyethylene glycol (PEG), with acarbohydrate, and/or with acyclic polyacetals derived frompolysaccharides (Papisov, 2001, ACS Symposium Series, 786:301). Certainembodiments may be made using the general teachings of U.S. Pat. No.5,543,158 to Gref et al., or WO publication WO2009/051837 by Von Andrianet al.

In some embodiments, polymers may be modified with a lipid or fatty acidgroup. In some embodiments, a fatty acid group may be one or more ofbutyric, caproic, caprylic, capric, lauric, myristic, palmitic, stearic,arachidic, behenic, or lignoceric acid. In some embodiments, a fattyacid group may be one or more of palmitoleic, oleic, vaccenic, linoleic,alpha-linoleic, gamma-linoleic, arachidonic, gadoleic, arachidonic,eicosapentaenoic, docosahexaenoic, or erucic acid.

In some embodiments, polymers may be polyesters, including copolymerscomprising lactic acid and glycolic acid units, such as poly(lacticacid-co-glycolic acid) and poly(lactide-co-glycolide), collectivelyreferred to herein as “PLGA”; and homopolymers comprising glycolic acidunits, referred to herein as “PGA,” and lactic acid units, such aspoly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid,poly-L-lactide, poly-D-lactide, and poly-D,L-lactide, collectivelyreferred to herein as “PLA.” In some embodiments, exemplary polyestersinclude, for example, polyhydroxyacids; PEG copolymers and copolymers oflactide and glycolide (e.g., PLA-PEG copolymers, PGA-PEG copolymers,PLGA-PEG copolymers, and derivatives thereof. In some embodiments,polyesters include, for example, poly(caprolactone),poly(caprolactone)-PEG copolymers, poly(L-lactide-co-L-lysine),poly(serine ester), poly(4-hydroxy-L-proline ester),poly[α-(4-aminobutyl)-L-glycolic acid], and derivatives thereof.

In some embodiments, a polymer may be PLGA. PLGA is a biocompatible andbiodegradable co-polymer of lactic acid and glycolic acid, and variousforms of PLGA are characterized by the ratio of lactic acid:glycolicacid. Lactic acid can be L-lactic acid, D-lactic acid, or D,L-lacticacid. The degradation rate of PLGA can be adjusted by altering thelactic acid:glycolic acid ratio. In some embodiments, PLGA to be used inaccordance with the present invention is characterized by a lacticacid:glycolic acid ratio of approximately 85:15, approximately 75:25,approximately 60:40, approximately 50:50, approximately 40:60,approximately 25:75, or approximately 15:85.

In some embodiments, polymers may be one or more acrylic polymers. Incertain embodiments, acrylic polymers include, for example, acrylic acidand methacrylic acid copolymers, methyl methacrylate copolymers,ethoxyethyl methacrylates, cyanoethyl methacrylate, aminoalkylmethacrylate copolymer, poly(acrylic acid), poly(methacrylic acid),methacrylic acid alkylamide copolymer, poly(methyl methacrylate),poly(methacrylic acid anhydride), methyl methacrylate, polymethacrylate,poly(methyl methacrylate) copolymer, polyacrylamide, aminoalkylmethacrylate copolymer, glycidyl methacrylate copolymers,polycyanoacrylates, and combinations comprising one or more of theforegoing polymers. The acrylic polymer may comprise fully-polymerizedcopolymers of acrylic and methacrylic acid esters with a low content ofquaternary ammonium groups.

In some embodiments, polymers can be cationic polymers. In general,cationic polymers are able to condense and/or protect negatively chargedstrands of nucleic acids (e.g. DNA, or derivatives thereof).Amine-containing polymers such as poly(lysine) (Zauner et al., 1998,Adv. Drug Del. Rev., 30:97; and Kabanov et al., 1995, BioconjugateChem., 6:7), poly(ethylene imine) (PEI; Boussif et al., 1995, Proc.Natl. Acad. Sci., USA, 1995, 92:7297), and poly(amidoamine) dendrimers(Kukowska-Latallo et al., 1996, Proc. Natl. Acad. Sci., USA, 93:4897;Tang et al., 1996, Bioconjugate Chem., 7:703; and Haensler et al., 1993,Bioconjugate Chem., 4:372) are positively-charged at physiological pH,form ion pairs with nucleic acids, and mediate transfection in a varietyof cell lines. In embodiments, the inventive synthetic nanocarriers maynot comprise (or may exclude) cationic polymers.

In some embodiments, polymers can be degradable polyesters bearingcationic side chains (Putnam et al., 1999, Macromolecules, 32:3658;Barrera et al., 1993, J. Am. Chem. Soc., 115:11010; Kwon et al., 1989,Macromolecules, 22:3250; Lim et al., 1999, J. Am. Chem. Soc., 121:5633;and Zhou et al., 1990, Macromolecules, 23:3399). Examples of thesepolyesters include poly(L-lactide-co-L-lysine) (Barrera et al., 1993, J.Am. Chem. Soc., 115:11010), poly(serine ester) (Zhou et al., 1990,Macromolecules, 23:3399), poly(4-hydroxy-L-proline ester) (Putnam etal., 1999, Macromolecules, 32:3658; and Lim et al., 1999, J. Am. Chem.Soc., 121:5633), and poly(4-hydroxy-L-proline ester) (Putnam et al.,1999, Macromolecules, 32:3658; and Lim et al., 1999, J. Am. Chem. Soc.,121:5633).

The properties of these and other polymers and methods for preparingthem are well known in the art (see, for example, U.S. Pat. Nos.6,123,727; 5,804,178; 5,770,417; 5,736,372; 5,716,404; 6,095,148;5,837,752; 5,902,599; 5,696,175; 5,514,378; 5,512,600; 5,399,665;5,019,379; 5,010,167; 4,806,621; 4,638,045; and 4,946,929; Wang et al.,2001, J. Am. Chem. Soc., 123:9480; Lim et al., 2001, J. Am. Chem. Soc.,123:2460; Langer, 2000, Acc. Chem. Res., 33:94; Langer, 1999, J.Control. Release, 62:7; and Uhrich et al., 1999, Chem. Rev., 99:3181).More generally, a variety of methods for synthesizing certain suitablepolymers are described in Concise Encyclopedia of Polymer Science andPolymeric Amines and Ammonium Salts, Ed. by Goethals, Pergamon Press,1980; Principles of Polymerization by Odian, John Wiley & Sons, FourthEdition, 2004; Contemporary Polymer Chemistry by Allcock et al.,Prentice-Hall, 1981; Deming et al., 1997, Nature, 390:386; and in U.S.Pat. Nos. 6,506,577, 6,632,922, 6,686,446, and 6,818,732.

In some embodiments, polymers can be linear or branched polymers. Insome embodiments, polymers can be dendrimers. In some embodiments,polymers can be substantially cross-linked to one another. In someembodiments, polymers can be substantially free of cross-links. In someembodiments, polymers can be used in accordance with the presentinvention without undergoing a cross-linking step. It is further to beunderstood that inventive synthetic nanocarriers may comprise blockcopolymers, graft copolymers, blends, mixtures, and/or adducts of any ofthe foregoing and other polymers. Those skilled in the art willrecognize that the polymers listed herein represent an exemplary, notcomprehensive, list of polymers that can be of use in accordance withthe present invention.

In some embodiments, the synthetic nanocarriers comprise one or morepolymers. The polymeric synthetic nanocarriers, therefore, can alsoinclude those described in WO publication WO2009/051837 by Von Andrianet al., including, but not limited to those, with one or morehydrophilic components. Preferably, the one or more polymers comprise apolyester, such as a poly(lactic acid), poly(glycolic acid),poly(lactic-co-glycolic acid), or polycaprolactone. More preferably, theone or more polymers comprise or further comprise a polyester coupled toa hydrophilic polymer, such as a polyether. In embodiments, thepolyether comprises polyethylene glycol. Still more preferably, the oneor more polymers comprise a polyester and a polyester coupled to ahydrophilic polymer, such as a polyether. In other embodiments, the oneor more polymers are coupled to one or more antigens and/or one or moreadjuvants. In embodiments, at least some of the polymers are coupled tothe antigen(s) and/or at least some of the polymers are coupled to theadjuvant(s). Preferably, when there are more than one type of polymer,one of the types of polymer is coupled to the antigen(s). Inembodiments, one of the other types of polymer is coupled to theadjuvant(s). For example, in embodiments, when the nanocarriers comprisea polyester and a polyester coupled to a hydrophilic polymer, such as apolyether, the polyester is coupled to the adjuvant, while the polyestercoupled to the hydrophilic polymer, such as a polyether, is coupled tothe antigen(s). In embodiments, where the nanocarriers comprise auniversal T cell antigen, such as a T helper cell antigen, the universalT cell antigen can be encapsulated in the nanocarrier.

In some embodiments, synthetic nanocarriers do not comprise a polymericcomponent. In some embodiments, synthetic nanocarriers may comprisemetal particles, quantum dots, ceramic particles, etc. In someembodiments, a non-polymeric synthetic nanocarrier is an aggregate ofnon-polymeric components, such as an aggregate of metal atoms (e.g.,gold atoms).

In some embodiments, synthetic nanocarriers may optionally comprise oneor more amphiphilic entities. In some embodiments, an amphiphilic entitycan promote the production of synthetic nanocarriers with increasedstability, improved uniformity, or increased viscosity. In someembodiments, amphiphilic entities can be associated with the interiorsurface of a lipid membrane (e.g., lipid bilayer, lipid monolayer,etc.). Many amphiphilic entities known in the art are suitable for usein making synthetic nanocarriers in accordance with the presentinvention. Such amphiphilic entities include, but are not limited to,phosphoglycerides; phosphatidylcholines; dipalmitoyl phosphatidylcholine(DPPC); dioleylphosphatidyl ethanolamine (DOPE);dioleyloxypropyltriethylammonium (DOTMA); dioleoylphosphatidylcholine;cholesterol; cholesterol ester; diacylglycerol; diacylglycerolsuccinate;diphosphatidyl glycerol (DPPG); hexanedecanol; fatty alcohols such aspolyethylene glycol (PEG); polyoxyethylene-9-lauryl ether; a surfaceactive fatty acid, such as palmitic acid or oleic acid; fatty acids;fatty acid monoglycerides; fatty acid diglycerides; fatty acid amides;sorbitan trioleate (Span®85) glycocholate; sorbitan monolaurate(Span®20); polysorbate 20 (Tween®20); polysorbate 60 (Tween®60);polysorbate 65 (Tween®65); polysorbate 80 (Tween®80); polysorbate 85(Tween®85); polyoxyethylene monostearate; surfactin; a poloxomer; asorbitan fatty acid ester such as sorbitan trioleate; lecithin;lysolecithin; phosphatidylserine; phosphatidylinositol;sphingomyelin;phosphatidylethanolamine (cephalin); cardiolipin; phosphatidic acid;cerebrosides; dicetylphosphate; dipalmitoylphosphatidylglycerol;stearylamine; dodecylamine; hexadecyl-amine; acetyl palmitate; glycerolricinoleate; hexadecyl sterate; isopropyl myristate; tyloxapol;poly(ethylene glycol)5000-phosphatidylethanolamine; poly(ethyleneglycol)400-monostearate; phospholipids; synthetic and/or naturaldetergents having high surfactant properties; deoxycholates;cyclodextrins; chaotropic salts; ion pairing agents; and combinationsthereof. An amphiphilic entity component may be a mixture of differentamphiphilic entities. Those skilled in the art will recognize that thisis an exemplary, not comprehensive, list of substances with surfactantactivity. Any amphiphilic entity may be used in the production ofsynthetic nanocarriers to be used in accordance with the presentinvention.

In some embodiments, synthetic nanocarriers may optionally comprise oneor more carbohydrates. Carbohydrates may be natural or synthetic. Acarbohydrate may be a derivatized natural carbohydrate. In certainembodiments, a carbohydrate comprises monosaccharide or disaccharide,including but not limited to glucose, fructose, galactose, ribose,lactose, sucrose, maltose, trehalose, cellbiose, mannose, xylose,arabinose, glucoronic acid, galactoronic acid, mannuronic acid,glucosamine, galatosamine, and neuramic acid. In certain embodiments, acarbohydrate is a polysaccharide, including but not limited to pullulan,cellulose, microcrystalline cellulose, hydroxypropyl methylcellulose(HPMC), hydroxycellulose (HC), methylcellulose (MC), dextran,cyclodextran, glycogen, hydroxyethylstarch, carageenan, glycon, amylose,chitosan, N,O-carboxylmethylchitosan, algin and alginic acid, starch,chitin, inulin, konjac, glucommannan, pustulan, heparin, hyaluronicacid, curdlan, and xanthan. In embodiments, the inventive syntheticnanocarriers do not comprise (or specifically exclude) carbohydrates,such as a polysaccharide. In certain embodiments, the carbohydrate maycomprise a carbohydrate derivative such as a sugar alcohol, includingbut not limited to mannitol, sorbitol, xylitol, erythritol, maltitol,and lactitol.

Compositions according to the invention comprise inventive syntheticnanocarriers in combination with pharmaceutically acceptable excipients,such as preservatives, buffers, saline, or phosphate buffered saline.The compositions may be made using conventional pharmaceuticalmanufacturing and compounding techniques to arrive at useful dosageforms. In an embodiment, inventive synthetic nanocarriers are suspendedin sterile saline solution for injection together with a preservative.

In embodiments, when preparing synthetic nanocarriers as carriers forantigens and/or adjuvants for use in vaccines, methods for coupling theantigens and/or adjuvants to the synthetic nanocarriers may be useful.If the antigen and/or adjuvant is a small molecule it may be ofadvantage to attach the antigen and/or adjuvant to a polymer prior tothe assembly of the synthetic nanocarriers. In embodiments, it may alsobe an advantage to prepare the synthetic nanocarriers with surfacegroups that are used to couple the antigen and/or adjuvant to thesynthetic nanocarrier through the use of these surface groups ratherthan attaching the antigen and/or adjuvant to a polymer and then usingthis polymer conjugate in the construction of synthetic nanocarriers.

Surface antigens can be coupled to the synthetic nanocarriers by avariety of methods. In embodiments, the surface antigen is coupled to anexternal surface of the synthetic nanocarrier covalently ornon-covalently.

In certain embodiments, the coupling can be via a covalent linker. Inembodiments, surface antigens and/or adjuvants according to theinvention can be covalently coupled to the external surface via a1,2,3-triazole linker formed by the 1,3-dipolar cycloaddition reactionof azido groups on the surface of the nanocarrier with surface antigensand/or adjuvants containing an alkyne group or by the 1,3-dipolarcycloaddition reaction of alkynes on the surface of the nanocarrier withsurface antigens and/or adjuvants containing an azido group. Suchcycloaddition reactions are preferably performed in the presence of aCu(I) catalyst along with a suitable Cu(I)-ligand and a reducing agentto reduce Cu(II) compound to catalytic active Cu(I) compound. ThisCu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) can also be referredas the click reaction.

Additionally, the covalent coupling may comprise a covalent linker thatcomprises an amide linker, a disulfide linker, a thioether linker, ahydrazone linker, a hydrazide linker, an imine or oxime linker, an ureaor thiourea linker, an amidine linker, an amine linker, and asulfonamide linker.

An amide linker is formed via an amide bond between an amine on onecomponent such as the peptide with the carboxylic acid group of a secondcomponent such as the nanocarrier. The amide bond in the linker can bemade using any of the conventional amide bond forming reactions withsuitably protected amino acids or peptides and activated carboxylic acidsuch N-hydroxysuccinimide-activated ester.

A disulfide linker is made via the formation of a disulfide (S—S) bondbetween two sulfur atoms of the form, for instance, of R₁—S—S—R₂. Adisulfide bond can be formed by thiol exchange of a surface antigensand/or adjuvants containing thiol/mercaptan group (—SH) with anotheractivated thiol group on a polymer or nanocarrier or a nanocarriercontaining thiol/mercaptan groups with an antigen and/or adjuvantscontaining activated thiol group.

A triazole linker, specifically a 1,2,3-triazole of the form

wherein R₁ and R₂ may be any chemical entities, is made by the1,3-dipolar cycloaddition reaction of an azide attached to a firstcomponent such as the nanocarrier with a terminal alkyne attached to asecond component such as the antigen and/or adjuvant. The 1,3-dipolarcycloaddition reaction is performed with or without a catalyst,preferably with Cu(I)-catalyst, which links the two components through a1,2,3-triazole function. This chemistry is described in detail bySharpless et al., Angew. Chem. Int. Ed. 41(14), 2596, (2002) and Meldal,et al, Chem. Rev., 2008, 108(8), 2952-3015 and is often referred to as“click” reaction or CuAAC.

In embodiments, a polymer containing an azide or alkyne group, terminalto the polymer chain is prepared. This polymer is then used to prepare asynthetic nanocarrier in such a manner that a plurality of the alkyne orazide groups are positioned on the surface of that nanocarrier.Alternatively, the synthetic nanocarrier can be prepared by anotherroute, and subsequently functionalized with alkyne or azide groups. Theantigen and/or adjuvant is prepared with the presence of either analkyne (if the polymer contains an azide) or an azide (if the polymercontains an alkyne) group. The antigen and/or adjuvant is then allowedto react with the nanocarrier via the 1,3-dipolar cycloaddition reactionwith or without a catalyst which covalently couples the antigen and/oradjuvant to the particle through the 1,4-disubstituted 1,2,3-triazolelinker.

A thioether linker is made by the formation of a sulfur-carbon(thioether) bond in the form, for instance, of R₁—S—R₂. Thioether can bemade by either alkylation of a thiol/mercaptan (—SH) group on onecomponent such as the antigen and/or adjuvant with an alkylating groupsuch as halide or epoxide on a second component such as the nanocarrier.Thioether linkers can also be formed by Michael addition of athiol/mercaptan group on one component such as an antigen and/oradjuvant to an electron-deficient alkene group on a second componentsuch as a polymer containing a maleimide group or vinyl sulfone group asthe Michael acceptor. In another way, thioether linkers can be preparedby the radical thiol-ene reaction of a thiol/mercaptan group on onecomponent such as an antigen and/or adjuvant with an alkene group on asecond component such as a polymer or nanocarrier.

A hydrazone linker is made by the reaction of a hydrazide group on onecomponent such as the antigen and/or adjuvant with an aldehyde/ketonegroup on the second component such as the nanocarrier.

A hydrazide linker is formed by the reaction of a hydrazine group on onecomponent such as the antigen and/or adjuvant with a carboxylic acidgroup on the second component such as the nanocarrier. Such reaction isgenerally performed using chemistry similar to the formation of amidebond where the carboxylic acid is activated with an activating reagent.

An imine or oxime linker is formed by the reaction of an amine orN-alkoxyamine (or aminooxy) group on one component such as the antigenand/or adjuvant with an aldehyde or ketone group on the second componentsuch as the nanocarrier.

An urea or thiourea linker is prepared by the reaction of an amine groupon one component such as the antigen and/or adjuvant with an isocyanateor thioisocyanate group on the second component such as the nanocarrier.

An amidine linker is prepared by the reaction of an amine group on onecomponent such as the antigen and/or adjuvant with an imidoester groupon the second component such as the nanocarrier.

An amine linker is made by the alkylation reaction of an amine group onone component such as the antigen and/or adjuvant with an alkylatinggroup such as halide, epoxide, or sulfonate ester group on the secondcomponent such as the nanocarrier. Alternatively, an amine linker canalso be made by reductive amination of an amine group on one componentsuch as the antigen and/or adjuvant with an aldehyde or ketone group onthe second component such as the nanocarrier with a suitable reducingreagent such as sodium cyanoborohydride or sodium triacetoxyborohydride.

A sulfonamide linker is made by the reaction of an amine group on onecomponent such as the antigen and/or adjuvant with a sulfonyl halide(such as sulfonyl chloride or sulfonyl fluoride) group on the secondcomponent such as the nanocarrier.

A sulfone linker is made by Michael addition of a nucleophile to a vinylsulfone. Either the vinyl sulfone or the nucleophile may be on thesurface of the nanoparticle or attached to the antigen or adjuvant.

The antigen or adjuvant can also be conjugated to the nanocarrier vianon-covalent conjugation methods. For examples, a negative chargedantigen or adjuvant can be conjugated to a positive charged nanocarrierthrough electrostatic adsorption. A antigen or adjuvant containing ametal ligand can also be conjugated to a nanocarrier containing a metalcomplex via a metal-ligand complex.

In embodiments, the antigen or adjuvant can be attached to a polymer,for example polylactic acid-block-polyethylene glycol, prior to theassembly of the synthetic nanocarrier or the synthetic nanocarrier canbe formed with reactive or activatible groups on its surface. In thelatter case, the antigen or adjuvant may be prepared with a group whichis compatible with the attachment chemistry that is presented by thesynthetic nanocarriers' surface. In other embodiments, a peptide antigencan be attached to VLPs or liposomes using a suitable linker. A linkeris a compound or reagent that capable of coupling two moleculestogether. In an embodiment, the linker can be a homobifuntional orheterobifunctional reagent as described in Hermanson 2008. For example,an VLP or liposome synthetic nanocarrier containing a carboxylic groupon the surface can be treated with a homobifunctional linker, adipicdihydrazide (ADH), in the presence of EDC to form the correspondingsynthetic nanocarrier with the ADH linker. The resulting ADH linkedsynthetic nanocarrier is then conjugated with a peptide antigen and/oradjuvant containing an acid group via the other end of the ADH linker onNC to produce the corresponding VLP or liposome peptide conjugate.

For detailed descriptions of available conjugation methods, seeHermanson G T “Bioconjugate Techniques”, 2nd Edition, Published byAcademic Press, Inc., 2008. In addition to covalent attachment theantigen and/or adjuvant can be coupled by adsorbtion to a pre-formedsynthetic nanocarrier or it/they can be coupled by encapsulation duringthe formation of the synthetic nanocarrier.

In embodiments, surface antigens can be non-covalently coupled tosynthetic nanocarriers using various non-covalent interactions includingbut not limited to charge interactions, affinity interactions, metalcoordination, physical adsorption, host-guest interactions, hydrophobicinteractions, TT stacking interactions, hydrogen bonding interactions,van der Waals interactions, magnetic interactions, electrostaticinteractions, dipole-dipole interactions, and/or combinations thereof.In embodiments, encapsulation is a form of coupling. When couplingcharged surface antigens, the synthetic nanocarriers can be produced inthe presence of surfactants which become adsorbed to surfaces of thesynthetic nanocarrier and in doing so they impart a charge to thesynthetic nanocarrier. Charged surface antigens can then benon-covalently attached to the charged synthetic nanocarrier by acharge-charge interaction (see for example O'Hagen WO2000006123A1).

In embodiments, the inventive synthetic nanocarriers can be combinedwith one or more adjuvants by admixing in the same vehicle or deliverysystem. Such adjuvants may include, but are not limited to the adjuvantprovided herein, such as mineral salts, such as alum, alum combined withmonphosphoryl lipid (MPL) A of Enterobacteria, such as Escherihia coli,Salmonella minnesota, Salmonella typhimurium, or Shigella flexneri orspecifically with MPL® (AS04), MPL A of above-mentioned bacteriaseparately, saponins, such as QS-21,Quil-A, ISCOMs, ISCOMATRIX™,emulsions such as MF59™, Montanide® ISA 51 and ISA 720, AS02(QS21+squalene+ MPL®), liposomes and liposomal formulations such asAS01, AS15, synthesized or specifically prepared microparticles andmicrocarriers such as bacteria-derived outer membrane vesicles (OMV) ofN. gonorrheae, Chlamydia trachomatis and others, or chitosan particles,depot-forming agents, such as Pluronic® block co-polymers, specificallymodified or prepared peptides, such as muramyl dipeptide, aminoalkylglucosaminide 4-phosphates, such as RC529, or proteins, such asbacterial toxoids or toxin fragments. The doses of such other adjuvantscan be determined using conventional dose ranging studies.

In embodiments, the inventive synthetic nanocarriers can be combinedwith other antigens different, similar or identical to those coupled toa nanocarrier (with or without adjuvant, utilizing or not utilizinganother delivery vehicle) administered separately at a differenttime-point and/or at a different body location and/or by a differentimmunization route or with another antigen and/or adjuvant-carryingsynthetic nanocarrier administered separately at a different time-pointand/or at a different body location and/or by a different immunizationroute.

Populations of synthetic nanocarriers may be combined to form dosageforms according to the present invention using traditionalpharmaceutical mixing methods. These include liquid-liquid mixing inwhich two or more suspensions, each containing one or more subset ofnanocarriers, are directly combined or are brought together via one ormore vessels containing diluent. As synthetic nanocarriers may also beproduced or stored in a powder form, dry powder-powder mixing could beperformed as could the re-suspension of two or more powders in a commonmedia. Depending on the properties of the nanocarriers and theirinteraction potentials, there may be advantages conferred to one oranother route of mixing.

Typical inventive compositions that comprise synthetic nanocarriers maycomprise inorganic or organic buffers (e.g., sodium or potassium saltsof phosphate, carbonate, acetate, or citrate) and pH adjustment agents(e.g., hydrochloric acid, sodium or potassium hydroxide, salts ofcitrate or acetate, amino acids and their salts) antioxidants (e.g.,ascorbic acid, alpha-tocopherol), surfactants (e.g., polysorbate 20,polysorbate 80, polyoxyethylene9-10 nonyl phenol, sodium desoxycholate),solution and/or cryo/lyo stabilizers (e.g., sucrose, lactose, mannitol,trehalose), osmotic adjustment agents (e.g., salts or sugars),antibacterial agents (e.g., benzoic acid, phenol, gentamicin),antifoaming agents (e.g., polydimethylsilozone), preservatives (e.g.,thimerosal, 2-phenoxyethanol, EDTA), polymeric stabilizers andviscosity-adjustment agents (e.g., polyvinylpyrrolidone, poloxamer 488,carboxymethylcellulose) and co-solvents (e.g., glycerol, polyethyleneglycol, ethanol).

Compositions according to the invention comprise inventive syntheticnanocarriers in combination with pharmaceutically acceptable excipientsor carriers. The compositions may be made using conventionalpharmaceutical manufacturing and compounding techniques to arrive atuseful dosage forms. Techniques suitable for use in practicing thepresent invention may be found in Handbook of Industrial Mixing: Scienceand Practice, Edited by Edward L. Paul, Victor A. Atiemo-Obeng, andSuzanne M. Kresta, 2004 John Wiley & Sons, Inc.; and Pharmaceutics: TheScience of Dosage Form Design, 2nd Ed. Edited by M. E. Auten, 2001,Churchill Livingstone. In an embodiment, inventive syntheticnanocarriers are suspended in sterile saline solution for injectiontogether with a preservative.

Doses of dosage forms contain varying amounts of populations ofsynthetic nanocarriers according to the invention. The amount ofsynthetic nanocarriers present in the inventive dosage forms can bevaried according to the nature of the sets of surface antigens, thetherapeutic benefit to be accomplished, and other such parameters. Inembodiments, dose ranging studies can be conducted to establish optimaltherapeutic amount of synthetic nanocarriers to be present in the dosageform. In embodiments, first and second populations are present in anamount effective to generate an immune response to the first set ofsurface antigens and the second set of surface antigens uponadministration to a subject. It may be possible to determine amounts ofthe first, second, and/or subsequent populations effective to generatean immune response using conventional dose ranging studies andtechniques in subjects. Inventive dosage forms may be administered at avariety of frequencies. In a preferred embodiment, at least oneadministration of the dosage form is sufficient to generate apharmacologically relevant response. In more preferred embodiment, atleast two administrations, at least three administrations, or at leastfour administrations, of the dosage form are utilized to ensure apharmacologically relevant response.

It is to be understood that the compositions of the invention can bemade in any suitable manner, and the invention is in no way limited tocompositions that can be produced using the methods described herein.Selection of an appropriate method may require attention to theproperties of the particular moieties being associated. In embodiments,methods of manufacture comprise preparing a first population ofsynthetic nanocarriers that comprise a first set of surface antigens;preparing a second population of synthetic nanocarriers that comprise asecond set of surface antigens; and combining the first and secondpopulations of synthetic nanocarriers into a pharmaceutical dosage form;wherein the first set of surface antigens and the second set of surfaceantigens are structurally or immunologically different.

In some embodiments, inventive synthetic nanocarriers are manufacturedunder sterile conditions or are terminally sterilized. This can ensurethat resulting composition are sterile and non-infectious, thusimproving safety when compared to non-sterile compositions. Thisprovides a valuable safety measure, especially when subjects receivingsynthetic nanocarriers have immune defects, are suffering frominfection, and/or are susceptible to infection. In some embodiments,inventive synthetic nanocarriers may be lyophilized and stored insuspension or as lyophilized powder depending on the formulationstrategy for extended periods without losing activity.

The inventive compositions may be administered by a variety of routes ofadministration, including but not limited to parenteral (such assubcutaneous, intramuscular, intravenous, or intradermal); oral;transnasal, intranasal, transmucosal, sublingual, rectal, ophthalmic,transdermal, transcutaneous or by a combination of these routes.

The compositions and methods described herein can be used to induce,enhance, modulate, stimulate, suppress, direct or redirect an immuneresponse. The compositions and methods described herein can be used inthe diagnosis, prophylaxis and/or treatment of conditions such ascancers, infectious diseases, metabolic diseases, degenerative diseases,autoimmune diseases, inflammatory diseases, immunological diseases, orother disorders and/or conditions. The compositions and methodsdescribed herein can also be used for the prophylaxis or treatment of anaddiction, such as an addiction to nicotine or a narcotic. Thecompositions and methods described herein can also be used for theprophylaxis and/or treatment of a condition resulting from the exposureto a toxin, hazardous substance, environmental toxin, or other harmfulagent.

The subjects provided herein can have or be at risk of having anaddiction to an abused or addictive substance.

The subjects provided herein can have or be at risk of having cancer.Cancers include, but are not limited to, breast cancer; biliary tractcancer; bladder cancer; brain cancer including glioblastomas andmedulloblastomas; cervical cancer; choriocarcinoma; colon cancer;endometrial cancer; esophageal cancer; gastric cancer; hematologicalneoplasms including acute lymphocytic and myelogenous leukemia, e.g., BCell CLL; T-cell acute lymphoblastic leukemia/lymphoma; hairy cellleukemia; chronic myelogenous leukemia, multiple myeloma;AIDS-associated leukemias and adult T-cell leukemia/lymphoma;intraepithelial neoplasms including Bowen's disease and Paget's disease;liver cancer; lung cancer; lymphomas including Hodgkin's disease andlymphocytic lymphomas; neuroblastomas; oral cancer including squamouscell carcinoma; ovarian cancer including those arising from epithelialcells, stromal cells, germ cells and mesenchymal cells; pancreaticcancer; prostate cancer; rectal cancer; sarcomas includingleiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, andosteosarcoma; skin cancer including melanoma, Merkel cell carcinoma,Kaposi's sarcoma, basal cell carcinoma, and squamous cell cancer;testicular cancer including germinal tumors such as seminoma,non-seminoma (teratomas, choriocarcinomas), stromal tumors, and germcell tumors; thyroid cancer including thyroid adenocarcinoma andmedullar carcinoma; and renal cancer including adenocarcinoma and Wilmstumor.

The subjects provided herein can have or be at risk of having aninfection or infectious disease. Infections or infectious diseasesinclude, but are not limited to, viral infectious diseases, such asAIDS, Chickenpox (Varicella), Common cold, Cytomegalovirus Infection,Colorado tick fever, Dengue fever, Ebola hemorrhagic fever, Hand, footand mouth disease, Hepatitis, Herpes simplex, Herpes zoster, HPV,Influenza (Flu), Lassa fever, Measles, Marburg hemorrhagic fever,Infectious mononucleosis, Mumps, Norovirus, Poliomyelitis, Progressivemultifocal leukencephalopathy, Rabies, Rubella, SARS, Smallpox(Variola), Viral encephalitis, Viral gastroenteritis, Viral meningitis,Viral pneumonia, West Nile disease and Yellow fever; bacterialinfectious diseases, such as Anthrax, Bacterial Meningitis, Botulism,Brucellosis, Campylobacteriosis, Cat Scratch Disease, Cholera,Diphtheria, Epidemic Typhus, Gonorrhea, Impetigo, Legionellosis, Leprosy(Hansen's Disease), Leptospirosis, Listeriosis, Lyme disease,Melioidosis, Rheumatic Fever, MRSA infection, Nocardiosis, Pertussis(Whooping Cough), Plague, Pneumococcal pneumonia, Psittacosis, Q fever,Rocky Mountain Spotted Fever (RMSF), Salmonellosis, Scarlet Fever,Shigellosis, Syphilis, Tetanus, Trachoma, Tuberculosis, Tularemia,Typhoid Fever, Typhus and Urinary Tract Infections; parasitic infectiousdiseases, such as African trypanosomiasis, Amebiasis, Ascariasis,Babesiosis, Chagas Disease, Clonorchiasis, Cryptosporidiosis,Cysticercosis, Diphyllobothriasis, Dracunculiasis, Echinococcosis,Enterobiasis, Fascioliasis, Fasciolopsiasis, Filariasis, Free-livingamebic infection, Giardiasis, Gnathostomiasis, Hymenolepiasis,Isosporiasis, Kala-azar, Leishmaniasis, Malaria, Metagonimiasis,Myiasis, Onchocerciasis, Pediculosis, Pinworm Infection, Scabies,Schistosomiasis, Taeniasis, Toxocariasis, Toxoplasmosis, Trichinellosis,Trichinosis, Trichuriasis, Trichomoniasis and Trypanosomiasis; fungalinfectious disease, such as Aspergillosis, Blastomycosis, Candidiasis,Coccidioidomycosis, Cryptococcosis, Histoplasmosis, Tinea pedis(Athlete's Foot) and Tinea cruris; prion infectious diseases, such asAlpers' disease, Fatal Familial Insomnia, Gerstmann-Sträussler-Scheinkersyndrome, Kuru and Variant Creutzfeldt-Jakob disease.

EXAMPLES

The invention will be more readily understood by reference to thefollowing examples, which are included merely for purposes ofillustration of certain aspects and embodiments of the present inventionand not as limitations.

Those skilled in the art will appreciate that various adaptations andmodifications of the just-described embodiments can be configuredwithout departing from the scope and spirit of the invention. Othersuitable techniques and methods known in the art can be applied innumerous specific modalities by one skilled in the art and in light ofthe description of the present invention described herein.

Therefore, it is to be understood that the invention can be practicedother than as specifically described herein. The above description isintended to be illustrative, and not restrictive. Many other embodimentswill be apparent to those of skill in the art upon reviewing the abovedescription. The scope of the invention should, therefore, be determinedwith reference to the appended claims, along with the full scope ofequivalents to which such claims are entitled.

Example 1 Formulation for First Population of Nanocarriers (Prophetic)

Synthetic nanocarriers comprising PLGA-R848 conjugate (adjuvant),PLA-PEG-N3 conjugate (linker to peptide antigen) and ova peptide (T-cellantigen) are prepared via a double emulsion method wherein the ovapeptide is encapsulated in the synthetic nanocarriers. To a suspensionof the synthetic nanocarriers (10 mg/mL in PBS (pH 7.4 buffer), 5 mL,containing about 12.5 mg (MW: 20, 000; 0.000625 mmol) of PLA-PEG-N3) isadded HPV L1-peptide comprising an acetylene linker (33 mg) under gentlestirring. A solution of sodium ascorbate (100 mM in H2O, 0.3 mL) isadded, followed by CuSO4 solution (10 mM in water, 0.6 mL). Theresulting light yellow suspension is stirred at 20 C for 15 h andadditional CuSO4 solution (0.3 mL) and sodium ascorbate solution (0.15mL) are added. The suspension is stirred for 5 h at 20 C and dilutedwith PBS buffer (pH 7.4) to 10 mL and is centrifuged to remove thesupernatant. The residual nanocarrier pellets are washed twice with PBSbuffer. The washed NCs are then re-suspended in 5 mL of PBS buffer andstored frozen. The conjugation of L1 peptide on the surface of thesynthetic nanocarriers is confirmed by HPLC analysis of the digestedsynthetic nanocarriers and by bioassay.

Example 2 Formulation for Second Population of Nanocarriers (Prophetic)

Using the general procedures outlines in Example 1 above, syntheticnanocarriers comprising PLA-R848, PLA-PEG-N3 and encapsulated ovapeptide are prepared and conjugated with an HPV L2 peptide to provide L2peptide conjugated synthetic nanocarriers.

Example 3 Formulation Combining First and Second Populations ofNanocarriers (Prophetic)

The synthetic nanocarrier preparations from Examples 1 and 2 above arethawed and diluted in PBS to a final concentration of 5 mg ofnanocarriers per milliliter. Equal aliquots of each (0.5 mL) arecombined to provide a population of nanocarriers that contain both theHPV L1 and L2 peptides.

Example 4 Preparations of Nanocarriers Preparation of NC-Nic-OVA

PLGA-R848, poly-D/L-lactide-co-glycolide,4-amino-2-(ethoxymethyl)-α,α-dimethyl-1H-imidazo[4,5-c]quinoline-1-ethanolamide of approximately 7,000 Da made from PLGA of 3:1 lactide toglycolide ratio and having approximately 8.5% w/w conjugated resiquimodcontent was custom manufactured at Princeton Global Synthesis (300George Patterson Drive #206, Bristol, Pa. 19007.) PLA-PEG-Nicotine(S-642), poly-D/L lactide-block-poly(ethyleneglycol)-(±)-trans-3′-hydroxymethylnicotine ether with PEG block ofapproximately 5,000 Da and PLA block of approximately 21,000 Da wascustom manufactured at Princeton Global Synthesis (300 George PattersonDrive #206, Bristol, Pa. 19007.) PLA-PEG-Maleimide, block co-polymerconsisting of a poly-D/L-lactide (PLA) block of approximately 22000 Daand a polyethylene glycol (PEG) block of approximately 2900 Da that isterminated by a maleimide functional group was synthesized fromcommercial starting materials by generating the PLA block byring-opening polymerization of dl-lactide with HO-PEG-Maleimide withdl-lactide. Polyvinyl alcohol PhEur, USP (85-89% hydrolyzed, viscosityof 3.4-4.6 mPa·s) was purchased from EMD Chemicals Inc. (480 SouthDemocrat Road Gibbstown, N.J. 08027. Part Number 4-88).

Solutions were prepared as follows:

Solution 1: 0.13N HCl in purified water.

Solution 2: PLGA-R848 @ 50 mg/mL, PLA-PEG-Nicotine @ 25 mg/mL, andPLA-PEG-Maleimide @ 25 mg/mL in dichloromethane was prepared bydissolving each polymer separately in dichloromethane at 100 mg/mL thencombining 2 parts PLGA-R848 solution to 1 part each PLA-PEG-Nicotinesolution and PLA-PEG-Maleimide solution.

Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM in 100 mM phosphatebuffer, pH 8.

Solution 4: 70 mM phosphate buffer, pH 8.

A primary (W1/O) emulsion was first created using Solution 1 andSolution 2. Solution 1 (0.2 mL) and Solution 2 (1.0 mL) were combined ina small glass pressure tube and sonicated at 50% amplitude for 40seconds using a Branson Digital Sonifier 250. A secondary (W1/O/W2)emulsion was then formed by adding Solution 3 (2.0 mL) to the primaryemulsion, vortexing to create a course dispersion, and then sonicatingat 30% amplitude for 40 seconds using the Branson Digital Sonifier 250.

The secondary emulsion was added to an open 50 mL beaker containing 70mM phosphate buffer solution (30 mL) and stirred at room temperature for2 hours to allow the dichloromethane to evaporate and the nanocarriersto form in suspension. A portion of the suspended nanocarriers waswashed by transferring the nanocarrier suspension to a centrifuge tube,spinning at 21,000 rcf for 45 minutes, removing the supernatant, andre-suspending the pellet in phosphate buffered saline. This washingprocedure was repeated and then the pellet was re-suspended in phosphatebuffered saline to achieve a nanocarrier suspension having a nominalconcentration of 10 mg/mL on a polymer basis. The nanocarrier suspensionwas stored frozen at −20 C until further use.

TABLE 4 NC-Nic-OVA Characterization Effective TLR Agonist, T-cellagonist, Nanocarrier Diameter (nm) % w/w % w/w NC-Nic-OVA 215 R848, 4.2None

-   -   (1) NC with PEG-Nicotine and PEG-MAL on the surface, prepared as        above; 6.5 mg/mL suspension in PBS buffer.    -   (2) OVA protein (Ovalbumin from egg white): Worthington, Lot#        POK12101, MW: 46000.    -   (3) Traut's reagent (2-iminothiolane.HCl): MP Biomedical,        Lot#8830KA, MW: 137.6    -   (4) pH 8 buffer (sodium phosphate, 20 mM with 0.5 mM EDTA).    -   (5) pH 7 1× PBS buffer.        OVA protein (10 mg) was dissolved in 1 mL pH 8 buffer. A freshly        made solution of Traut's reagent in pH 8 buffer (0.25 mL, 2        mg/mL) was added to the OVA protein solution. The resulting        solution was stirred under argon in the dark for 1.5 h. The        solution was diafiltered with MWCO 3K diafilter tube and washed        with pH 8 buffer twice. The resulting modified OVA with thiol        group was dissolved in 1 mL pH 8 buffer under argon. The NC        suspension (3 mL, 6.5 mg/mL) was centrifuged to remove the        supernatant. The modified OVA solution was then mixed with the        NC pellets. The resulting suspension was stirred at rt under        argon in the dark for 12 h. The NC suspension was then diluted        to 10 mL with pH 7 PBS and centrifuged. The resulting NC was        pellet washed with 2×10 mL pH 7 PBS. The NC-Nic-OVA conjugates        were then resuspended in pH 7 PBS (ca. 6 mg/mL, 3 mL) stored at        4° C.

Preparation of NC-OVA

PLGA-R848, poly-D/L-lactide-co-glycolide,4-amino-2-(ethoxymethyl)-α,α-dimethyl-1H-imidazo[4,5-c]quinoline-1-ethanolamide of approximately 7,000 Da made from PLGA of 3:1 lactide toglycolide ratio and having approximately 8.5% w/w conjugated resiquimodcontent was custom manufactured at Princeton Global Synthesis (300George Patterson Drive #206, Bristol, Pa. 19007.) PLA-PEG-Maleimide,block co-polymer consisting of a poly-D/L-lactide (PLA) block ofapproximately 22000 Da and a polyethylene glycol (PEG) block ofapproximately 2900 Da that is terminated by a maleimide functional groupwas synthesized from commercial starting materials by generating the PLAblock by ring-opening polymerization of dl-lactide withHO-PEG-Maleimide. Polyvinyl alcohol PhEur, USP (85-89% hydrolyzed,viscosity of 3.4-4.6 mPa·s) was purchased from EMD Chemicals Inc. (480South Democrat Road Gibbstown, N.J. 08027. Part Number 4-88).

Solutions were prepared as follows:

Solution 1: 0.13N HCl in purified water.

Solution 2: PLGA-R848 @ 50 mg/mL and PLA-PEG-Maleimide @ 50 mg/mL indichloromethane was prepared by dissolving each polymer separately indichloromethane at 100 mg/mL then combining 1 part PLGA-R848 solution to1 part PLA-PEG-Maleimide solution.

Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM in 100 mM phosphatebuffer, pH 8.

Solution 4: 70 mM phosphate buffer, pH 8.

A primary (W1/O) emulsion was first created using Solution 1 andSolution 2. Solution 1 (0.2 mL) and Solution 2 (1.0 mL) were combined ina small glass pressure tube and sonicated at 50% amplitude for 40seconds using a Branson Digital Sonifier 250. A secondary (W1/O/W2)emulsion was then formed by adding Solution 3 (2.0 mL) to the primaryemulsion, vortexing to create a course dispersion, and then sonicatingat 30% amplitude for 40 seconds using the Branson Digital Sonifier 250.

The secondary emulsion was added to an open 50 mL beaker containing 70mM phosphate buffer solution (30 mL) and stirred at room temperature for2 hours to allow the dichloromethane to evaporate and the nanocarriersto form in suspension. A portion of the suspended nanocarriers waswashed by transferring the nanocarrier suspension to a centrifuge tube,spinning at 21,000 rcf for 45 minutes, removing the supernatant, andre-suspending the pellet in phosphate buffered saline. This washingprocedure was repeated, and then the pellet was re-suspended inphosphate buffered saline to achieve a nanocarrier suspension having anominal concentration of 10 mg/mL on a polymer basis. The nanocarriersuspension was stored frozen at −20° C. until further use.

TABLE 5 NC-OVA Characterization Effective TLR Agonist, T-cell agonist,Nanocarrier Diameter (nm) % w/w % w/w NC-OVA 208 R848, 4.3 None

-   -   (1) NC with PEG-MAL on the surface, prepared as above; 6 mg/mL        suspension in PBS buffer.    -   (2) OVA protein (Ovalbumin from egg white): Worthington,        Lot#POK12101, MW: 46000.    -   (3) Traut's reagent (2-iminothiolane.HCl): MP Biomedical, Lot#        8830KA, MW: 137.6.    -   (4) pH 8 buffer (sodium phosphate, 20 mM with 0.5 mM EDTA).    -   (5) pH 7 1× PBS buffer.

OVA protein (20 mg) was dissolved in 1 mL pH 8 buffer. A freshly madesolution of Traut's reagent in pH 8 buffer (0.5 mL, 2 mg/mL) was addedto the OVA protein solution. The resulting solution was stirred underargon in the dark for 1.5 h. The solution was diafiltered with MWCO 3Kdiafilter tube and washed with pH 8 buffer twice. The resulting modifiedOVA with thiol group was dissolved in 1 mL pH 8 buffer under argon. TheNC suspension (4 mL, 6 mg/mL) was centrifuged to remove the supernatant.The modified OVA solution was then mixed with the NC pellets. Theresulting suspension was stirred at rt under argon in the dark for 12 h.The NC suspension was then diluted to 10 mL with pH 7 PBS andcentrifuged. The resulting NC was pellet washed with 2×10 mL pH 7 PBS.The NC-OVA conjugates were then resuspended in pH 7 PBS (ca. 6 mg/mL, 4mL) stored at 4° C.

Preparation of NC-HA5

Ovalbumin peptide 323-339 amide acetate salt, was purchased from BachemAmericas Inc. (3132 Kashiwa Street, Torrance Calif. 90505. Product code4065609.) PLGA-R848, poly-D/L-lactide-co-glycolide,4-amino-2-(ethoxymethyl)-α,α-dimethyl-1H-imidazo[4,5-c]quinoline-1-ethanolamide of approximately 7,000 Da made from PLGA of 3:1 lactide toglycolide ratio and having approximately 8.5% w/w conjugated resiquimodcontent was custom manufactured at Princeton Global Synthesis (300George Patterson Drive #206, Bristol, Pa. 19007.) PLA-PEG-Maleimide,block co-polymer consisting of a poly-D/L-lactide (PLA) block ofapproximately 22000 Da and a polyethylene glycol (PEG) block ofapproximately 2900 Da that is terminated by a maleimide functionalgroup, was synthesized from commercial starting materials by generatingthe PLA block by ring-opening polymerization of dl-lactide withHO-PEG-Maleimide. Polyvinyl alcohol PhEur, USP (85-89% hydrolyzed,viscosity of 3.4-4.6 mPa·s) was purchased from EMD Chemicals Inc. (480South Democrat Road Gibbstown, N.J. 08027. Part Number 4-88).

Solutions were prepared as follows:

Solution 1: Ovalbumin peptide 323-339 @ 20 mg/mL was prepared in 0.13NHCl at room temperature.

Solution 2: PLGA-R848 @ 50 mg/mL and PLA-PEG-Maleimide @ 50 mg/mL indichloromethane was prepared by dissolving each polymer separately indichloromethane at 100 mg/mL then combining 1 part PLGA-R848 solution to1 part PLA-PEG-Maleimide solution.

Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM in 100 mM phosphatebuffer, pH 8.

Solution 4: 70 mM phosphate buffer, pH 8.

A primary (W1/O) emulsion was first created using Solution 1 andSolution 2. Solution 1 (0.2 mL) and Solution 2 (1.0 mL) were combined ina small glass pressure tube and sonicated at 50% amplitude for 40seconds using a Branson Digital Sonifier 250. A secondary (W1/O/W2)emulsion was then formed by adding Solution 3 (2.0 mL) to the primaryemulsion, vortexing to create a course dispersion, and then sonicatingat 30% amplitude for 40 seconds using the Branson Digital Sonifier 250.

The secondary emulsion was added to an open 50 mL beaker containing 70mM phosphate buffer solution (30 mL) and stirred at room temperature for2 hours to allow the dichloromethane to evaporate and the nanocarriersto form in suspension. A portion of the suspended nanocarriers waswashed by transferring the nanocarrier suspension to a centrifuge tube,spinning at 21,000 rcf for 45 minutes, removing the supernatant, andre-suspending the pellet in phosphate buffered saline. This washingprocedure was repeated, and then the pellet was re-suspended inphosphate buffered saline to achieve a nanocarrier suspension having anominal concentration of 10 mg/mL on a polymer basis. The nanocarriersuspension was stored frozen at −20° C. until further use.

TABLE 6 NC-HA5 Characterization Effective TLR Agonist, T-cell agonist,Nanocarrier Diameter (nm) % w/w % w/w NC-HA5 216 R848, 3.6 Ova peptide323-339, 2.0

-   -   (1) NC with PEG-MAL on the surface, prepared as above; 6.7 mg/mL        suspension in PBS buffer.    -   (2) HAS protein: Recombinant Hemagglutinin, A/Vietnam/1203/2004,        MW: 72000, supplied as a solution in pH 7 PBS-tween buffer (0.55        mg/mL).    -   (3) Traut's reagent (2-iminothiolane.HCl): MP Biomedical, Lot#        8830KA, MW: 137.6.    -   (4) pH 8 buffer (sodium phosphate, 20 mM with 0.5 mM EDTA).    -   (5) pH 7 1× PBS buffer.

HA5 protein (0.21 g in 0.38 mL pH 7.1 PBS-tween buffer) was diluted to0.5 mL with pH 8 buffer. A freshly made solution of Traut's reagent inpH 8 buffer (0.02 mL, 2 mg/mL) was added to the HA5 protein solution.The resulting solution was stirred under argon in the dark for 1.5 h.The solution was diafiltered with MWCO 3K diafilter tube and washed withpH 8 buffer twice. The resulting modified HAS protein with thiol groupwas dissolved in 0.5 mL pH 8 buffer under argon. The NC suspension (3mL, 6.7 mg/mL) was centrifuged to remove the supernatant. The modifiedHAS solution was then mixed with the NC pellets. The resultingsuspension was stirred at rt under argon in the dark for 12 h. The NCsuspension was then diluted to 10 mL with pH 7 PBS and centrifuged. Theresulting NC was pellet washed with 2×10 mL pH 7 PBS. The NC-HA5conjugates were then resuspended in pH 7 PBS (ca. 6 mg/mL, 3 mL) storedat 4° C.

Preparation of NC-L2, NC-M2e or NC-M2e-L2

Ovalbumin peptide 323-339 amide acetate salt, was purchased from BachemAmericas Inc. (3132 Kashiwa Street, Torrance Calif. 90505. Product code4065609.) PLGA-R848, poly-D/L-lactide-co-glycolide,4-amino-2-(ethoxymethyl)-α,α-dimethyl-1H-imidazo[4,5-c]quinoline-1-ethanolamide of approximately 7,000 Da made from PLGA of 3:1 lactide toglycolide ratio and having approximately 8.5% w/w conjugated resiquimodcontent was custom manufactured at Princeton Global Synthesis (300George Patterson Drive #206, Bristol, Pa. 19007.) PLA-PEG-C6-N₃, blockco-polymer consisting of a poly-D/L-lactide (PLA) block of approximately23000 Da and a polyethylene glycol (PEG) block of approximately 2000 Dathat is terminated by an amide-conjugated C₆H₁₂ linker to an azide, wassynthesized by conjugating HO-PEG-COOH to an amino-C₆H₁₂-azide and thengenerating the PLA block by ring-opening polymerization of the resultingHO-PEG-C6-N3 with dl-lactide. Polyvinyl alcohol PhEur, USP (85-89%hydrolyzed, viscosity of 3.4-4.6 mPa·s) was purchased from EMD ChemicalsInc. (480 South Democrat Road Gibbstown, N.J. 08027. Part Number 4-88).

Solutions were prepared as follows:

Solution 1: Ovalbumin peptide 323-339 @ 20 mg/mL was prepared in 0.13NHCl at room temperature.

Solution 2: PLGA-R848 @ 50 mg/mL and PLA-PEG-C6-N₃ @ 50 mg/mL indichloromethane was prepared by dissolving each separately at 100 mg/mLin dichloromethane then combining in equal parts by volume.

Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM in 100 mM phosphatebuffer, pH 8.

Solution 4: 70 mM phosphate buffer, pH 8.

A primary (W1/O) emulsion was first created using Solution 1 andSolution 2. Solution 1 (0.2 mL) and Solution 2 (1.0 mL) were combined ina small glass pressure tube and sonicated at 50% amplitude for 40seconds using a Branson Digital Sonifier 250. A secondary (W1/O/W2)emulsion was then formed by adding Solution 3 (2.0 mL) to the primaryemulsion, vortexing to create a course dispersion, and then sonicatingat 30% amplitude for 40 seconds using the Branson Digital Sonifier 250.

The secondary emulsion was added to an open 50 mL beaker containing 70mM phosphate buffer solution (30 mL) and stirred at room temperature for2 hours to allow the dichloromethane to evaporate and the nanocarriersto form in suspension. A portion of the suspended nanocarriers waswashed by transferring the nanocarrier suspension to a centrifuge tube,spinning at 21,000 rcf for 45 minutes, removing the supernatant, andre-suspending the pellet in phosphate buffered saline. This washingprocedure was repeated, and then the pellet was re-suspended inphosphate buffered saline to achieve a nanocarrier suspension having anominal concentration of 10 mg/mL on a polymer basis. Two identicalbatches were created and then combined to form a single homogenoussuspension at which was stored frozen at −20° C. until further use.

TABLE 7 NC-L2, NC-M2e or NC-M2e-L2 Characterization Effective TLRAgonist, Antigen, Nanocarrier Diameter (nm) % w/w % w/w NC-L2, NC- 209R848, 4.2 Ova 323-339 peptide, 2.4 M2e or NC- M2e-L2

-   -   (1) Nanocarriers with surface PEG-C6-N3 containing PLGA-R848 and        Ova-peptide, prepared as above, 7 mg/mL suspension in PBS.    -   (2) HPV16 L2 peptide modified with an alkyne linker attached to        C-terminal Lys amino group; Bachem Americas, Inc, Lot B06055, MW        2595, TFA salt; Sequence:        H-Ala-Thr-Gln-Leu-Tyr-Lys-Thr-Cys-Lys-Gln-Ala-Gly-Thr-Cys-Pro-Pro-Asp-Ile-Ile-Pro-Lys-Val-Lys(5-hexynoyl)-NH2(with        Cys-Cys disulfide bond).    -   (3) Catalysts: CuSO4 , 100 mM in DI water; THPTA ligand, 200 mM        in DI water; sodium ascorbate, 200 mM in DI water freshly        prepared.    -   (4) pH 7.4 PBS buffer.

The NC suspension (7 mg/mL, 4 mL) was concentrated to ca. 1 mL in volumeby centrifugation. A solution of L2 peptide (20 mg) in 2 mL PBS bufferwas added. A pre-mixed solution of 0.2 mL of CuSO4 (100 mM) and 0.2 mLof THPTA ligand (200 mM) was added, followed by 0.4 mL of sodiumascorbate (200 mM). The resulting light yellow suspension was stirred indark at ambient room temperature for 18 h. The suspension was thendiluted with PBS buffer to 10 mL and centrifuged to remove thesupernatant. The NC-L2 conjugates were further pellet washed twice with10 mL PBS buffer and resuspended in pH 7.4 buffer at final concentrationof ca. 6 mg/mL (ca. 4 mL) and stored at 4° C.

-   -   (1) Nanocarriers with surface PEG-C6-N3 containing PLGA-R848 and        Ova-peptide, prepared as above, 7 mg/mL suspension in PBS.    -   (2) M2e peptide modified with an alkyne linker attached to        C-terminal Gly; CS Bio Co, Catalog No. CS4956, Lot: H308, MW        2650, TFA salt; Sequence:        H-Met-Ser-Leu-Leu-Thr-Glu-Val-Glu-Thr-Pro-Thy-Arg-Asn-Glu-Trp-Glu-Cys-Arg-Cys-Ser-Asp-Gly-Gly-NHCH2CCH.    -   (3) Catalysts: CuSO4, 100 mM in DI water; THPTA ligand, 200 mM        in DI water; sodium ascorbate, 200 mM in DI water freshly        prepared.    -   (4) pH 7.4 PBS buffer.

The NC suspension (7 mg/mL, 4 mL) was concentrated to ca. 1 mL in volumeby centrifugation. A solution of M2e peptide (20 mg) in 2 mL PBS bufferwas added. A pre-mixed solution of 0.2 mL of CuSO4 (100 mM) and 0.2 mLof THPTA ligand (200 mM) was added, followed by 0.4 mL of sodiumascorbate (200 mM). The resulting light yellow suspension was stirred indark at ambient room temperature for 18 h. The suspension was thendiluted with PBS buffer to 10 mL and centrifuged to remove thesupernatant. The NC-M2e conjugates were further pellet washed twice with10 mL PBS buffer and resuspended in pH 7.4 buffer at final concentrationof ca. 6 mg/mL (ca. 4 mL) and stored at 4° C.

-   -   (1) Nanocarriers with surface PEG-C6-N3 containing PLGA-R848 and        Ova-peptide, prepared as above, 7 mg/mL suspension in PBS.    -   (2) HPV16 L2 peptide modified with an alkyne linker attached to        C-terminal Lys amino group; Bachem Americas, Inc, Lot B06055, MW        2595, TFA salt; Sequence:        H-Ala-Thr-Gln-Leu-Tyr-Lys-Thr-Cys-Lys-Gln-Ala-Gly-Thr-Cys-Pro-Pro-Asp-Ile-Ile-Pro-Lys-Val-Lys(5-hexynoyl)-NH2(with        Cys-Cys disulfide bond).    -   (3) M2e peptide modified with an alkyne linker attached to        C-terminal Gly; CS Bio Co, Catalog No. CS4956, Lot: H308, MW        2650, TFA salt; Sequence:        H-Met-Ser-Leu-Leu-Thr-Glu-Val-Glu-Thr-Pro-Thy-Arg-Asn-Glu-Trp-Glu-Cys-Arg-Cys-Ser-Asp-Gly-Gly-NHCH2CCH.    -   (4) Catalysts: CuSO4, 100 mM in DI water; THPTA ligand, 200 mM        in DI water; sodium ascorbate, 200 mM in DI water freshly        prepared.    -   (5) pH 7.4 PBS buffer.

The NC suspension (7 mg/mL, 2 mL) was concentrated to ca. 0.5 mL involume by centrifugation. A mixture of L2 peptide (5 mg) and M2e peptide(5 mg) in 1 mL PBS buffer was added. A pre-mixed solution of 0.2 mL ofCuSO4 (100 mM) and 0.2 mL of THPTA ligand (200 mM) was added, followedby 0.4 mL of sodium ascorbate (200 mM). The resulting light yellowsuspension was stirred in dark at ambient room temperature for 18 h. Thesuspension was then diluted with PBS buffer to 10 mL and centrifuged toremove the supernatant. The NC-M2e-L2 conjugates were further pelletwashed twice with 10 mL PBS buffer and resuspended in pH 7.4 buffer atfinal concentration of ca. 6 mg/mL (ca. 2 mL) and stored at 4° C.

Example 5 Immunization with Two Monovalent Antigen Nanocarriers Leads toImmune Response to Both Antigens

Anti-nicotine (dark gray bars) and anti-ovalbumin (light gray bars)antibody titers in unimmunized mice and mice injected with NC-Nic andNC-OVA (as prepared in Example 4) (5 animals/group; s.c., 100 μg of eachNC per injection, 2 times at 3-wk intervals) were measured. Titers forday 33 after immunization with NC are shown in FIG. 1 (ELISA againstpolylysine-nicotine or ovalbumin protein) (group 1: unimmunized; group2: immunized with NC-Nic and NC-OVA).

Mice were injected with 100 μg of NC-Nic (nanocarrier exhibitingnicotine on the outer surface and containing OP-II helper peptide andR848 adjuvant in the NC) and 100 μg of NC-OVA (nanocarrier exhibitingovalbumin on the outer surface and containing OP-II helper peptide andR848 adjuvant in the NC) (subcutaneously, hind limbs) at 3-weekintervals (days 0 and 21). Serum anti-nicotine and anti-ovalbuminantibody titers were measured at day 33 after immunization with NC.Anti-nicotine and anti-ovalbumin antibody titers (EC₅₀) as measured byELISA against polylysine-nicotine or ovalbumin protein are shown (FIG.1). Titers for control unimmunized mice are also shown. The resultsdemonstrate that mice immunized with a combination of two monovalentantigen nanocarriers (NC-Nic and NC-OVA) generate antibodies to bothantigens.

Example 6 Immunization with Monovalent and Bivalent Antigen NanocarriersLead to Immune Response to All Three Antigens

Anti-nicotine, anti-ovalbumin, and anti-L2 peptide antibody titers inunimmunized mice and mice injected with NC-Nic-OVA and NC-L2 (asprepared in Example 4) (5 animals/group; s.c., 100 μg of each NC perinjection, 2 times at 3-wk intervals) were measured. Titers for day 33after immunization with NC are shown in FIG. 2 (ELISA againstpolylysine-nicotine, ovalbumin protein, or PLA-PEG-L2 peptide) (group 1:unimmunized; group 2: immunized with NC-Nic-OVA and NC-L2).

Mice were injected with 100 μg of NC-Nic-OVA (nanocarrier exhibitingnicotine and ovalbumin on the outer surface and containing OP-II helperpeptide and R848 adjuvant in the NC) and 100 μg of NC-L2 (nanocarrierexhibiting HPV L2 peptide (aa17-36) on the outer surface and containingOP-II helper peptide and R848 adjuvant in the NC) (subcutaneously, hindlimbs) at 3-week intervals (days 0 and 21). Serum anti-nicotine,anti-ovalbumin, and anti-L2 peptide antibody titers were measured at day33 after immunization with NC. Anti-nicotine, anti-ovalbumin, andanti-L2 peptide antibody titers (EC₅₀) as measured by ELISA againstpolylysine-nicotine, ovalbumin protein, and L2 peptide are shown (FIG.2). Titers for control unimmunized mice are also shown. The resultsdemonstrate that mice immunized with a combination of one monovalent andone bivalent antigen nanocarrier (NC-Nic-OVA and NC-L2) generateantibodies to all three antigens.

Example 7 Immunization with Two Bivalent Antigen Nanocarriers Leads toImmune Response to all Four Antigens

Anti-nicotine, anti-ovalbumin, anti-M2e peptide, and anti-L2 peptideantibody titers in unimmunized mice and mice injected with NC-Nic-OVAand NC-M2e-L2 (as prepared in Example 4) (5 animals/group; s.c., 100 μgof each NC per injection, 2 times at 3-wk intervals) were measured.Titers for day 33 after immunization with NC are shown in FIG. 3 (ELISAagainst polylysine-nicotine, ovalbumin protein, PLA-PEG-M2e peptide, orPLA-PEG-L2 peptide) (group 1: unimmunized; group 2: immunized withNC-Nic-OVA and NC-M2e-L2).

Mice were injected with 100 μg of NC-Nic-OVA (nanocarrier exhibitingnicotine and ovalbumin on the outer surface and containing OP-II helperpeptide and R848 adjuvant in the NC) and 100 μg of NC-M2e-L2(nanocarrier exhibiting influenza M2e peptide (aa2-27) and HPV L2peptide (aa17-36) on the outer surface and containing OP-II helperpeptide and R848 adjuvant in the NC) (subcutaneously, hind limbs) with3-week intervals (days 0 and 21). Serum anti-nicotine, anti-ovalbumin,anti-M2e peptide, and anti-L2 peptide antibody titers were measured atday 33 after immunization with NC. Anti-nicotine, anti-ovalbumin,anti-M2e peptide, and anti-L2 peptide antibody titers (EC₅₀) as measuredby ELISA against polylysine-nicotine, ovalbumin protein, M2e peptide,and L2 peptide are shown (FIG. 3). Titers for control unimmunized miceare also shown. The results demonstrate that mice immunized with acombination of two bivalent antigen nanocarriers (NC-Nic-OVA andNC-M2e-L2) generate antibodies to all four antigens.

Example 8 Immunization with Two Monovalent Peptide Antigen NanocarriersLeads to Immune Response to Both Peptide Antigens

Anti-M2e peptide and anti-L2 peptide antibody titers in unimmunized miceand mice injected with NC-M2e and NC-L2 (as prepared in Example 4) (5animals/group; s.c., 100 μg of each NC per injection, 2 times at 3-wkintervals) were measured. Titers for day 33 after immunization with NCare shown in FIG. 4 (ELISA against PLA-PEG-M2e peptide or PLA-PEG-L2peptide) (group 1: unimmunized; group 2: immunized with NC-M2e andNC-L2).

Mice were injected with 100 μg of NC-M2e (nanocarrier exhibitinginfluenza M2e peptide (aa2-27) on the outer surface and containing OP-IIhelper peptide and R848 adjuvant in the NC) and 100 μg of NC-L2(nanocarrier exhibiting HPV L2 peptide (aa17-36) on the outer surfaceand containing OP-II helper peptide and R848 adjuvant in the NC)(subcutaneously, hind limbs) at 3-week intervals (days 0 and 21). Serumanti-M2e peptide and anti-L2 peptide antibody titers were measured atday 33 after immunization with NC. Anti-M2e peptide and anti-L2 peptideantibody titers (EC₅₀) as measured by ELISA against M2e peptide and L2peptide are shown (FIG. 4). Titers for control unimmunized mice are alsoshown. These results demonstrate that mice immunized with a combinationof two monovalent peptide antigen nanocarriers (NC-M2e and NC-L2)generate antibodies to both peptide antigens.

Example 9 Immunization with Two Monovalent Protein Antigen NanocarriersLeads to Immune Response to Both Protein Antigens

Anti-HA5 protein and anti-ovalbumin protein antibody titers inunimmunized mice and mice injected with NC-HA5 and NC-OVA (as preparedin Example 4) (5 animals/group; s.c., 100 μg of each NC per injection, 2times with 3-wk intervals) were measured. Titers for day 33 afterimmunization with NC are shown in FIG. 5 (ELISA against H5N1 HA proteinor ovalbumin protein) (group 1: unimmunized; group 2: immunized withNC-HA5 and NC-OVA).

Mice were injected with 100 μg of NC-HA5 protein (nanocarrier exhibitinginfluenza H5N1 HA protein on the outer surface and containing OP-IIhelper peptide and R848 adjuvant in the NC) and 100 μg of NC-OVA(nanocarrier exhibiting ovalbumin on the outer surface and containingOP-II helper peptide and R848 adjuvant in the NC) (subcutaneously, hindlimbs) at 3-week intervals (days 0 and 21). Serum anti-HA5 andanti-ovalbumin antibody titers were measured at day 33 afterimmunization with NC. Anti-HA5 and anti-ovalbumin antibody titers (EC₅₀)as measured by ELISA against H5N1 HA protein and ovalbumin protein areshown (FIG. 5). Titers for control unimmunized mice are also shown.These results demonstrate that mice immunized with a combination of twomonovalent protein antigen nanocarriers (NC-HA5 and NC-OVA) generateantibodies to both protein antigens.

Example 10 Immunization with Two Monovalent and One Bivalent AntigenNanocarriers Leads to Immune Response to all Four Antigens

Anti-HA, anti-ovalbumin, anti-M2e peptide, and anti-L2 peptide antibodytiters in unimmunized mice and mice injected with NC-HA5, NC-OVA, andNC-M2e-L2 (as prepared in Example 4) (5 animals/group; s.c., 100 μg ofeach NC per injection, 2 times at 3-wk intervals) were measured. Titersfor day 33 after immunization with NC are shown in FIG. 6 (ELISA againstHA protein, ovalbumin protein, PLA-PEG-M2e peptide, or PLA-PEG-L2peptide) (group 1: unimmunized; group 2: immunized with NC-HA5, NC-OVA,and NC-M2e-L2).

Mice were injected with 100 μg of NC-HA5 protein (nanocarrier exhibitinginfluenza H5N1 HA protein on the outer surface and containing OP-IIhelper peptide and R848 adjuvant in the NC), 100 μg of NC-OVA(nanocarrier exhibiting ovalbumin on the outer surface and containingOP-II helper peptide and R848 adjuvant in the NC), and 100 μg ofNC-M2e-L2 (nanocarrier exhibiting influenza M2e peptide (aa2-27) and HPVL2 peptide (aa17-36) on the outer surface and containing OP-II helperpeptide and R848 adjuvant in the NC) (subcutaneously, hind limbs) at3-week intervals (days 0 and 21). Serum anti-HA, anti-ovalbumin,anti-M2e peptide, and anti-L2 peptide antibody titers were measured atday 33 after immunization with NC. Anti-HA, anti-ovalbumin, anti-M2epeptide, and anti-L2 peptide antibody titers (EC₅₀) as measured by ELISAagainst HA protein, ovalbumin protein, M2e peptide, and L2 peptide areshown (FIG. 6). Titers for control unimmunized mice are also shown.These results demonstrate that mice immunized with a combination of twomonovalent and one bivalent antigen nanocarrier (NC-HA5, NC-OVA, andNC-M2e-L2) generate antibodies to all four antigens.

Example 11 Immunization with Two Monovalent and One Bivalent AntigenNanocarriers Leads to Immune Response to all Four Antigens

Antibody titers in mice immunized with a combination of NC-M2e, NC-L2peptide and NC-nicotine-ovalbumin (as prepared in Example 4) weremeasured. NC-M2e and NC-L2 peptide contained OP-II T-helper peptide(2.0% and 2.4%, correspondingly) and R848 adjuvant (3.6% and 4.3%,correspondingly); NC-nicotine-ovalbumin contained R848 adjuvant (4.2%).Each bar of FIG. 7 represents the titer against antigen. Five animalsper group were immunized s.c. with 120 μg of each NC per injection, 2times at 3-wk intervals. Titers for day 33 after the first immunizationare shown (ELISA done against PLA-PEG-M2e, PLA-PEG-L2, ovalbumin andpolylysine-nicotine, correspondingly).

These results demonstrate that immunization with a combination of twoNCs each carrying a different peptide antigen together with a NCcarrying another two antigens results in generation of antibodies to allfour NC-carried antigens. When identical amounts of three NC, the firstcontaining surface M2e peptide from influenza A virus (ectodomain of M2matrix protein, amino acids 2-27), the second containing surface L2peptide from HPV virus (amino acids 17-36 from L2 capsid protein ofHPV-16), and the third carrying surface nicotine and ovalbumin proteinwere used for animal immunization, a strong humoral response was inducedin all animals against all four NC-coupled antigens (FIG. 7). Noreactivity was detected in the sera of preimmune mice.

Example 12 Immunization with Two Monovalent Nanocarriers with Antigen inDifferent Steric Orientations Leads to Immune Response both OrientationsPreparation of NC-3′-Nicotine

Ovalbumin peptide 323-339 amide acetate salt, was purchased from BachemAmericas Inc. (3132 Kashiwa Street, Torrance Calif. 90505. Product code4065609.) PLGA-R848, poly-D/L-lactide-co-glycolide,4-amino-2-(ethoxymethyl)-α,α-dimethyl-1H-imidazo[4,5-c]quinoline-1-ethanolamide of approximately 7,000 Da made from PLGA of 3:1 lactide toglycolide ratio and having approximately 8.5% w/w conjugated resiquimodcontent was custom manufactured at Princeton Global Synthesis (300George Patterson Drive #206, Bristol, Pa. 19007.) PLA-PEG-Nicotine(S-642), poly-D/L lactide-block-poly(ethyleneglycol)-(±)-trans-3′-hydroxymethylnicotine ether with PEG block ofapproximately 5,000 Da and PLA block of approximately 21,000 Da wascustom manufactured at Princeton Global Synthesis (300 George PattersonDrive #206, Bristol, Pa. 19007.) Polyvinyl alcohol PhEur, USP (85-89%hydrolyzed, viscosity of 3.4-4.6 mPa·s) was purchased from EMD ChemicalsInc. (480 South Democrat Road Gibbstown, N.J. 08027. Part Number 4-88).

Solutions were prepared as follows:

Solution 1: Ovalbumin peptide 323-339 @ 20 mg/mL was prepared in 0.13NHCl at room temperature.

Solution 2: PLGA-R848 @ 50 mg/mL, PLA-PEG-Nicotine @ 25 mg/mL, and PLA @25 mg/mL in dichloromethane were prepared by dissolving each polymerseparately in dichloromethane at 100 mg/mL then combining 2 partsPLGA-R848 solution to 1 part each PLA-PEG-Nicotine solution and PLAsolution.

Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM in 100 mM phosphatebuffer, pH 8.

Solution 4: 70 mM phosphate buffer, pH 8.

A primary (W1/O) emulsion was first created using Solution 1 andSolution 2. Solution 1 (0.2 mL) and Solution 2 (1.0 mL) were combined ina small glass pressure tube and sonicated at 50% amplitude for 40seconds using a Branson Digital Sonifier 250. A secondary (W1/O/W2)emulsion was then formed by adding Solution 3 (2.0 mL) to the primaryemulsion, vortexing to create a course dispersion, and then sonicatingat 30% amplitude for 40 seconds using the Branson Digital Sonifier 250.

The secondary emulsion was added to an open 50 mL beaker containing 70mM phosphate buffer solution (30 mL) and stirred at room temperature for2 hours to allow the dichloromethane to evaporate and the nanocarriersto form in suspension. A portion of the suspended nanocarriers waswashed by transferring the nanocarrier suspension to a centrifuge tube,spinning at 21,000 rcf for 45 minutes, removing the supernatant, andre-suspending the pellet in phosphate buffered saline. This washingprocedure was repeated, and then the pellet was re-suspended inphosphate buffered saline to achieve a nanocarrier suspension having anominal concentration of 10 mg/mL on a polymer basis. The nanocarriersuspension was stored frozen at −20° C. until further use.

TABLE 8 NC-3′-Nicotine Characterization Effective TLR Agonist, T-cellagonist, Nanocarrier Diameter (nm) % w/w % w/w NC-3′- 193 R848, 4.2 Ova323-339 peptide, 2.1 Nicotine

Preparation of NC-1′-Nicotine

Ovalbumin peptide 323-339 amide acetate salt, was purchased from BachemAmericas Inc. (3132 Kashiwa Street, Torrance Calif. 90505. Product code4065609.) PLGA-R848, poly-D/L-lactide-co-glycolide,4-amino-2-(ethoxymethyl)-α,α-dimethyl-1H-imidazo[4,5-c]quinoline-1-ethanolamide of approximately 7,000 Da made from PLGA of 3:1 lactide toglycolide ratio and having approximately 8.5% w/w conjugated resiquimodcontent was custom manufactured at Princeton Global Synthesis (300George Patterson Drive #206, Bristol, Pa. 19007.) PLA-PEG-1′-Nic, ablock co-polymer consisting of a poly-D/L-lactide (PLA) block ofapproximately 23000 Da and a polyethylene glycol (PEG) block ofapproximately 2000 Da that is conjugated to nicotine via a 4-carbonlinkage to the 1′ amino group on nicotine was synthesized. In brief,nicotine with a butyl-alcohol linker at the 1′ position was made intoHO-PEG-1′-Nic by polymerization with ethylene oxide, and the PLAextension was then generated by ring-opening polymerization of theHO-PEG-1′-Nic with dl-lactide. PLA with an inherent viscosity of 0.22dL/g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive,Birmingham, Ala. 35211. Product Code 100 DL 2A.) Polyvinyl alcoholPhEur, USP (85-89% hydrolyzed, viscosity of 3.4-4.6 mPa·s) was purchasedfrom EMD Chemicals Inc. (480 South Democrat Road Gibbstown, N.J. 08027.Part Number 4-88).

Solutions were prepared as follows:

Solution 1: Ovalbumin peptide 323-339 @ 20 mg/mL was prepared in 0.13NHCl at room temperature.

Solution 2: PLGA-R848 @ 50 mg/mL, PLA-PEG-1′-Nic @ 25 mg/mL, and PLA @25 mg/mL in dichloromethane was prepared by dissolving each polymerseparately in dichloromethane at 100 mg/mL then combining 2 partsPLGA-R848 solution to 1 part each PLA-PEG-1′-Nic solution and PLAsolution.

Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM in 100 mM phosphatebuffer, pH 8.

Solution 4: 70 mM phosphate buffer, pH 8.

A primary (W1/O) emulsion was first created using Solution 1 andSolution 2. Solution 1 (0.2 mL) and Solution 2 (1.0 mL) were combined ina small glass pressure tube and sonicated at 50% amplitude for 40seconds using a Branson Digital Sonifier 250. A secondary (W1/O/W2)emulsion was then formed by adding Solution 3 (2.0 mL) to the primaryemulsion, vortexing to create a course dispersion, and then sonicatingat 30% amplitude for 60 seconds using the Branson Digital Sonifier 250.

The secondary emulsion was added to an open 50 mL beaker containing 70mM phosphate buffer solution (30 mL) and stirred at room temperature for2 hours to allow the dichloromethane to evaporate and the nanocarriersto form in suspension. A portion of the suspended nanocarriers waswashed by transferring the nanocarrier suspension to a centrifuge tube,spinning at 21,000 rcf for 45 minutes, removing the supernatant, andre-suspending the pellet in phosphate buffered saline. This washingprocedure was repeated, and then the pellet was re-suspended inphosphate buffered saline to achieve a nanocarrier suspension having anominal concentration of 10 mg/mL on a polymer basis. The nanocarriersuspension was stored frozen at −20° C. until further use.

TABLE 9 NC-1′-Nicotine Characterization Effective TLR Agonist, T-cellagonist, Nanocarrier Diameter (nm) % w/w % w/w NC-1′- 238 R848, 3.9 Ova323-339 peptide, 2.8 Nicotine

Immunization and Results

Antibody titers in mice immunized with a combination of NC-3′-nicotineand NC-1′-nicotine were measured. NC-3′-nicotine and NC-1′-nicotinecontained OP-II T-helper peptide (2.1%) and R848 adjuvant (4.2%). Eachbar of FIG. 8 represents the titer against antigen. Five animals pergroup were immunized s.c. with 120 μg of each NC per injection, 2 timesat 3-wk intervals. Titers for day 33 after the first immunization areshown (ELISA done against polylysine-nicotine, respectively).

These results show that immunization with a combination of two NCs eachcarrying the same antigen but in different steric orientations resultsin the generation of antibodies against both of these differentorientations of the same antigen. When identical amounts of two NCs, thefirst containing surface nicotine attached to NC in the 3′-position andthe second attached to NC in the 1′-position were used for animalimmunization, a strong humoral response was induced in all animalsagainst both orientations of nicotine (FIG. 8). No reactivity wasdetected in the sera of preimmune mice.

Example 13 Preparations of Polymers and Nanocarriers Preparation ofPLGA-R848

PLGA-R848 was prepared by reaction of PLGA polymer containing an acidend group with R848 in the presence of coupling agent such as HBTU asfollows. A mixture of PLGA (Lakeshores Polymers, MW ˜5000, 7525DLG1A,acid number 0.7 mmol/g, 10 g, 7.0 mmol) and HBTU (5.3 g, 14 mmol) inanhydrous EtOAc (160 mL) was stirred at room temperature under argon for50 minutes. Compound R848 (2.2 g, 7 mmol) was added, followed bydiisopropylethylamine (DIPEA) (5 mL, 28 mmol). The mixture was stirredat room temperature for 6 h and then at 50-55° C. overnight (about 16h). After cooling, the mixture was diluted with EtOAc (200 mL) andwashed with saturated NH4Cl solution (2×40 mL), water (40 mL) and brinesolution (40 mL). The solution was dried over Na2SO4 (20 g) andconcentrated to a gel-like residue. Isopropyl alcohol (IPA) (300 mL) wasthen added and the polymer conjugate precipitated out of solution. Thepolymer was then washed with IPA (4×50 mL) to remove residual reagentsand dried under vacuum at 35-40° C. for 3 days as a white powder (10.26g, MW by GPC is 5200, R848 loading is 12% by HPLC).

In a similar manner, PLA-R848 was prepared by the reaction of PLA-CO2H(polylactide with acid ending group) with R848.

Preparation of PLA-PEG-CO2H

A mixture of HO-PEG-CO2H (MW: 2000, 1.0 g, 0.5 mmol), dl-lactide (10.8g, 75 mmol) and Na2SO4 (15 g) in a 100 mL round bottom flask was driedunder vacuum at 60° C. for 2 days. Anhydrous toluene (30 ML) was added,and the mixture was heated to reflux under argon. Sn(Oct)2 (0.162 mL,0.5 mmol) was added. The mixture was refluxed under argon overnight andcooled to ambient room temperature. The mixture was diluted with CH2Cl2(200 mL) and filtered through a pad of Celite. The filtrate wasconcentrated to a dense sticky residue. 10% MeOH in diethyl ether (200mL) was added to precipitate out the polymer with vigorous stirring. Thepolymer was further washed with 10% MeOH in ether (100 mL) and driedunder vacuum at 30° C. to give the PLA-PEG-CO2H copolymer as anoff-white foamy solid (10.0 g, H NMR in CDCl3 showed the polymer has MWof 21000).

Preparation of PLA-PEG-NH2

A mixture of HO-PEG-NH2.HCl (MW: 3500, 1.0 g, 0.28 mmol), dl-lactide(6.1 g, 42 mmol) and Na2SO4 (10 g) in a 100 mL round bottom flask wasdried under vacuum at 60° C. for 1 day. Anhydrous toluene (30 ML) wasadded and the mixture was heated to 90° C. under argon. Sn(Oct)2 (0.1mL, 0.28 mmol) was added. The mixture was refluxed under argon overnightand cooled to ambient room temperature. The mixture was diluted withethyl acetate (200 mL) and filtered through a pad of Celite. Thefiltrate was concentrated to a dense sticky residue. 10% MeOH in t-butylmethyl ether (MTBE) (200 mL) was added to precipitate out the polymerwith vigorous stirring. The polymer was further washed with 5% MeOH inMTBE (50 mL) and MTBE (50 mL) and dried under vacuum at 30° C. to givethe PLA-PEG-NH2.HCl copolymer as an off-white foamy solid (5.0 g, H NMRin CDCl3 showed the polymer has MW of 18000).

Preparation of PLA-PEG-PEGS-N3

PLA-PEG-N3 polymer was prepared by ring opening polymerization ofHO-PEG-azide with dl-lactide in the presence of a catalyst such asSn(Oct)2 as follows. HO-PEG-CO2H (MW 3500, 1.33 g, 0.38 mmol) wastreated with NH2-PEG3-N3 (MW 218.2, 0.1 g, 0.458 mmol) in the presenceof DCC (MW 206, 0.117 g, 0.57 mmol) and NHS (MW 115, 0.066 g, 0.57 mmol)in dry DCM (10 mL) overnight. After filtration to remove insolublebyproduct (DCC-urea), the solution was concentrated and then dilutedwith ether to precipitate out the polymer, HO-PEG-PEG3-N3 (1.17 g).After drying, HO-PEG-PEG3-N3 (MW 3700, 1.17 g, 0.32 mmol) was mixed withdl-lactide (recrystallized from EtOAc, MW 144, 6.83 g, 47.4 mmol) andNa2SO4 (10 g) in a 100 mL flask. The solid mixture was dried undervacuum at 45° C. overnight and dry toluene (30 mL) was added. Theresulting suspension was heated to 110° C. under argon and Sn(Oct)2 (MW405, 0.1 mL, 0.32 mmol) was added. The mixture was heated at reflux for18 h and cooled to rt. The mixture was diluted with DCM (50 mL) andfiltered. After concentration to an oily residue, MTBE (200 mL) wasadded to precipitate out the polymer which was washed once with 100 mLof 10% MeOH in MTBE and 50 mL of MTBE. After drying, PLA-PEG-PEG3-N3 wasobtained as a white foam (7.2 g, average MW: 23,700 by H NMR).

Preparation of PLA-PEG-C6-N3

HO-PEG-CO2H (MW 3500, 1.00 g, 0.29 mmol) was treated with6-azido-1-hexylamine (H2N-C6-N3) (MW 142, 0.081 g, 0.57 mmol) in thepresence of DCC (MW 206, 0.118 g, 0.57 mmol) and NHS (MW 115, 0.066 g,0.57 mmol) in dry DCM (10 mL) overnight. After filtration to removeinsoluble byproduct (DCC-urea), the solution was concentrated and thendiluted with MTBE to precipitate out the polymer which was then washedtwice with MTBE and dried under vacuum at 30° C. overnight to giveHO-PEG-C6-N3 polymer (1.1 g). HO-PEG-C6-N3 polymer (1.1 g, 0.29 mmol)and dl-lactide (6.5 g, 45 mmol) were mixed in dry toluene (60 mL). Themixture was heated to reflux while 30 mL of toluene was removed byazeotrope distillation. The resulting solution was cooled to 100° C. andSn(Oct)2 (0.095 mL, 0.29 mmol) was added. The solution was heated atreflux under argon overnight and cooled to rt. The solution was thenadded to 150 mL of 2-propanol to precipitate out the polymer which waswashed with 2-propanol (100 mL) and dried under vacuum at 30° C. for 2days to give PLA-PEG-C6-N3 copolymer as an off-white solid (6.8 g, MW byGPC is 27000 with DPI of 1.5).

Preparation of PLA-PEG(5K)-CONH2NH2

A mixture of HO-PEG(5 k)-CO2H (JenKem Technology, USA) (MW: 5000, 1.0 g,0.2 mmol), tert-butyl carbazate (Boc-hydrazide) (MW: 132, 0.053 g, 0.4mmol), DCC (MW 206, 0.083 g, 0.4 mmol) and N-hydroxysuccinimide (NHS)(MW 115, 0.05 g, 0.4 mmol) in dry DCM (15 mL) was stirred at rt for 25h. The insoluble DCC-urea was removed by filtration and the filtrate wasconcentrated. The residual was added to 50 mL of MTBE to precipitate outthe polymer which was washed twice with 40 mL of MTBE and dried undervacuum for 2 days to give HO-PEG(5 k)-CONHNHtBoc as a white powder (1.07g). HO-PEG(5 k)-CONHNHtBoc polymer (1.07 g, 0.20 mmol) and dl-lactide(4.32 g, 30 mmol) were mixed in dry toluene (70 mL). The mixture washeated to reflux while 50 mL of toluene was removed by azeotropedistillation. The resulting solution was cooled to 100° C. and Sn(Oct)2(0.065 mL, 0.20 mmol) was added. The solution was heated at reflux underargon for 22 h and cooled to rt. The solution was then added to 150 mLof 2-propanol to precipitate out the polymer which was washed with2-propanol (60 mL) and dried under vacuum at 30° C. for 2 days to givePLA-PEG(5 k)-CONHNHtBoc copolymer as a white solid chunk. The polymerwas dissolved in 50 mL of dry DCM and cooled with ice water.Trifluoroacetic acid (TFA) (15 mL) was added and the resulting solutionwas stirred at rt overnight. The yellowish solution was concentrated todryness. The residual was added to 200 mL of 2-propanol to precipitateout the polymer which was washed with 100 mL of 2-propanol. The polymerwas dried at 30° C. under vacuum to give the desired polymer asPLA-PEG(5 k)-CONHNH2 (3.4 g, MW by NMR: 24000).

Preparation of PLA-PEG-MAL

HO-PEG(3K)-maleimide (HO-PEG-MAL) (Laysan Bio, Inc) (MW: 3000, 0.6 g,0.2 mmol) was mixed with dl-lactide (recrystallized from EtOAc, MW 144,4.32 g, 30 mmol) and Na2SO4 (4 g) in a 100 mL flask. The solid mixturewas dried under vacuum at 60° C. overnight and dry toluene (20 mL) wasadded. The resulting suspension was heated to 110° C. under argon andSn(Oct)2 (MW 405, 0.065 mL, 0.2 mmol) was added. The mixture was heatedat reflux for 20 h and cooled to rt. The mixture was diluted with DCM(50 mL) and filtered. After concentration to an oily residue, 10% MeOHin ethyl ether (80 mL) was added to precipitate out the polymer whichwas washed once with 80 mL of 10% MeOH in ether and 60 mL of ether.After drying at 30° C. under vacuum overnight, PLA-PEG(3K)-MAL wasobtained as a white foam (3.26 g, average MW: 24,000 by H NMR).

Preparation of PLA-PEG-SH (Prophetic)

PLA-PEG-SH copolymer is prepared according to the literature (Nisha C.Kalarickal, et al; Macromolecules 2007, 40:1874-1880). Briefly, thefollowing steps are performed.

Step-1. Preparation of tBuS-PEG: Anhydrous THF (22 mL), potassiumnaphthalene (0.2 M solution in THF, 12 mL), and tBu-SH (0.54 mL, 4.8mmol) are charged into a sealed 100 mL round-bottom flask. Thecomponents are stirred for at least 15 min to ensure the formation ofthiolates, at which point liquid ethylene oxide (EO) (11.5 mL, 0.230mol) is added using a two-headed needle. The polymerization reaction iscarried out for 48 h, and the product is recovered by precipitation incold diethyl ether. MW of the polymer by GPC is about 2100.

Step-2. Preparation of (PEG-S)2: tBu-S-PEG from Step-1 (1.0 g) isdissolved in DMSO (19 mL) followed by addition of TFA (106 mL, 15/85v/v) to a final polymer concentration of 8 mg/mL. The reaction isstirred for 20 min, after which TFA is removed by rotary evaporation.The residual is then precipitated twice in cold diethyl ether to recoverthe crude PEG disulfide. The crude (PEG-S)2 is further purified byfractional precipitation. Thus, the polymer (1.0 g) is dissolved indichloromethane (100 mL), and then cold diethyl ether is added stepwisewith stirring until the appearance of a precipitate. The solution isfurther stirred for 30 min, and the precipitated mass is isolated byfiltration and dried in vacuo. The recovery yield of PEG disulfide,(PEG-S)2, at the end of two to three fractional precipitations is in therange 55-60%.

Step-3. Preparation of (PLA-b-PEG-S)2 by ring-opening polymerization ofdl-lactide: (PEG-S)2 (0.4 g, 0.10 mmol) and dl-lactide (4.32 g, 30 mmol)are mixed in dry toluene (70 mL). The mixture is heated to reflux while50 mL of toluene is removed by azeotrope distillation. The resultingsolution was cooled to 100° C. and Sn(Oct)2 (0.065 mL, 0.20 mmol) wasadded. The solution is heated at reflux under argon for 18-20 h andcooled to rt. The solution is then added to 150 mL of 2-propanol toprecipitate out the polymer which is washed with 2-propanol (60 mL) andether (60 mL) and dried under vacuum at 30° C. for 2 days to give(PLA-PEG-S)2 (ca. 4.0 g, MW: 46000).

Step-4. Preparation of PLA-PEG-SH by reduction of (PLA-PEG-S)2: The(PLA-PEG-S)2 from Step-3 (3.2 g, 0.07 mmol) is dissolved in deoxygenatedTHF (25 mL), and Bu3P (1.7 mL, 7.0 mmol, 100 equiv with respect todisulfide units) is added. The reaction mixture is stirred under argonat room temperature overnight. The reduced thiolated polymer isrecovered by precipitation in cold diethyl ether followed by filtrationunder argon atmosphere and further dried under vacuum to give PLA-PEG-SHas an off white chunky solid (ca. 3.0, MW: 23000).

Preparation of Nanocarriers with Surface PEG-X Containing EncapsulatedOva Peptide

Nanocarriers comprising PLGA-R848, PLA-PEG-X (where X=carboxylic acid(CO2H), amine (NH2), C6-azide (C6-N3) or PEG3-azide (PEG3-N3), hydrazide(CONHNH2), maleimide (MAL), thiol (SH) and nitrilotriacetic acid group(NTA)) containing ova peptide were prepared via a double emulsion methodwherein the ova peptide was encapsulated in the nanocarriers. Polyvinylalcohol (Mw=11 KD −31 KD, 87-89% partially hydrolyzed) was purchasedfrom J T Baker. Ovalbumin peptide 323-339, (sequence:H-Ile-Ser-Gln-Ala-Val-His-Ala-Ala-His-Ala-Glu-Ile-Asn-Glu-Ala-Gly-Arg-NH2,acetate salt, Lot#B06395) was obtained from Bachem Americas Inc. (3132Kashiwa Street, Torrance Calif. 90505), PLA with acid end group(100DL2A) was obtained from SurModics Pharmaceuticals (756 Tom MartinDrive, Birmingham, Ala. 35211); PLGA-R848, and PLA-PEG-X conjugates wereprepared as described above in this same example.

The above materials were used to prepare the following solutions:

-   -   1. PLGA-R848 conjugate in methylene chloride @ 100 mg/mL,    -   2. PLA-PEG-X in methylene chloride @ 100 mg/mL,    -   3. PLA (100DL2A) in methylene chloride @ 100 mg/mL,    -   4. Ovalbumin peptide 323-339 in 0.13N HCl @ 70 mg/mL, and    -   5. Polyvinyl alcohol in 100 mM pH 8 phosphate buffer @50 mg/mL.

Solution #1 (0.50 mL), solution #2 (0.25 mL) and solution #3 (0.25 mL)were combined and solution #4 in 0.13N HCl (0.1mL) was added in a smallvessel and the mixture was sonicated at 50% amplitude for 40 secondsusing a Branson Digital Sonifier 250. To this emulsion was addedsolution # 5 (2.0 mL) and sonication at 30% amplitude for 40 secondsusing the Branson Digital Sonifier 250 was performed on the secondemulsion. This was then added to a stirring beaker containing a 70 mM pH8 phosphate buffer solution (30 mL), and this mixture was stirred atroom temperature for 2 hours to form the nanocarriers.

To wash the nanocarriers, a portion of the nanocarrier dispersion (26.5mL) was transferred to a 50 mL centrifuge tube and spun at 9500 rpm(13,800 g) for one hour at 4° C. The supernatant was removed, and thepellet was re-suspended in 26.5 mL of phosphate buffered saline. Thecentrifuge procedure was repeated and the pellet was re-suspended in 8.3g of phosphate buffered saline for a final nanocarrier dispersion ofabout 10 mg/mL containing encapsulated ova peptide.

Preparation of Nanocarriers with surface PEG-X Without Encapsulated OvaPeptide

In a similar manner to the procedure described immediately above,nanocarriers without ova peptide were prepared where solution #4 waseliminated in the preparation.

Example 14 Nanocarriers with Obtained Versus Derived Antigen (Prophetic)

Nanocarriers with PTH: Nanocarriers with surface PEG-CONHNH2 hydrazidegroups are prepared as described above in Example 13. PTH (parathyroidhormone) protein is acylated via the lysine amino group with4-formyl-benzoic acid in the presence of EDC. HCl and NHS are used togenerate PTH containing benzaldehyde groups. After purification viadialfiltration with a MWCO 1K filter, the modified PTH is conjugatedwith the NCs containing the hydrazide on the surface in PBS buffer (pH8-9). After purification by pellet washing with PBS buffer, theresulting NC-modified PTH conjugate is suspended in pH 7.4 buffer.

Nanocarriers with Modified PTH: Nanocarriers with surface PEG-CO2H groupare prepared as described above in Example 13. The NCs are thenactivated with excess EDC/NHS in pH 6 PBS buffer at 4° C. for 1-2 h. Theactivated NCs are then pellet washed with pH 6.0 buffer to removeun-reacted EDC/NHS. Modified PTH dissolved in the same PBS buffer isthen added to the resulting NC suspension. The conjugation is allowed toproceed at 4° C. overnight. After pellet washing with PBS buffer, theresulting NC-Modified PTH conjugate is suspended in pH 7.4 PBS buffer.

Equal portions of the two nanocarriers can then be combined to form a NCsuspension for further testing.

Example 15 Monovalent and Bivalent Nanocarriers with Antigens from theSame Genus of Infectious Agent (Prophetic)

Nanocarriers with surface PEG-CO2H groups are prepared as describedabove in Example 13. The NCs are then activated with excess EDC/NHS inpH 6 PBS buffer at 4° C. for 1-2 h. The activated NCs are then pelletwashed with pH 6.0 buffer to remove un-reacted EDC/NHS and suspended inpH 6.0 buffer. Human Influenza A virus HA protein trimer and HA M2eprotein dissolved in pH 6.0 buffer is then added to the resulting NCsuspension. The conjugation is allowed to proceed at 4° C. overnight.After pellet washing with PBS buffer, the resulting NC-HA proteintrimer/M2e protein conjugate is suspended in pH 7.4 PBS buffer.

In the same fashion, a NC-HA monomer protein conjugate is prepared usingmonomeric Human Influenza A virus HA protein.

The nanocarriers can then be combined to form a NC suspension forfurther testing.

Example 16 Monovalent and Bivalent Nanocarriers with Antigens from aDifferent Genus of Infectious Agent (Prophetic)

Nanocarriers with surface PEG-CO2H groups are prepared as describedabove in Example 13. The NCs are then activated with excess EDC/NHS inpH 6 PBS buffer at 4° C. for 1-2 h. The activated NCs are then pelletwashed with pH 6.0 buffer to remove un-reacted EDC/NHS and suspended inpH 6.0 buffer. Human Influenza A virus HA protein trimer and HA M2eprotein dissolved in pH 6.0 buffer is then added to the resulting NCsuspension. The conjugation is allowed to proceed at 4° C. overnight.After pellet washing with PBS buffer, the resulting NC-HA proteintrimer/M2e protein conjugate is suspended in pH 7.4 PBS buffer.

In the same fashion, a NC-infectious salmon anemia virus conjugate isprepared using inactivated infectious salmon anemia virus.

The nanocarriers can then be combined to form a NC suspension forfurther testing.

Example 17 Monovalent Nanocarriers with Antigens from the Same Speciesof Infectious Agent (Prophetic)

Nanocarriers with surface PEG-CO2H groups are prepared as describedabove in Example 13. The NCs are then activated with excess EDC/NHS inpH 6 PBS buffer at 4° C. for 1-2 h. The activated NCs are then pelletwashed with pH 6.0 buffer to remove un-reacted EDC/NHS and suspended inpH 6.0 buffer. Measles hemaglutinin antigen (a recombinant fragmentcontaining the measles hemagglutinin immunodominant regions, amino acids106-114 and 519-550) is dissolved in pH 6.0 buffer and then added to theresulting NC suspension. The conjugation is allowed to proceed at 4° C.overnight. After pellet washing with PBS buffer, the resultingNC-measles hemaglutinin conjugate is suspended in pH 7.4 PBS buffer.

In the same fashion, NC-measles fusion antigen conjugate is preparedusing a fragment of measles fusion protein (a recombinant fragmentcorresponding to amino acids 399-525 of measles large fusion protein).

The nanocarriers can then be combined to form a NC suspension forfurther testing.

Example 18 Monovalent Nanocarriers with Antigens from Different Speciesof Infectious Agent (Prophetic)

Nanocarriers with surface PEG-CO2H groups are prepared as describedabove in Example 13. The NCs are then activated with excess EDC/NHS inpH 6 PBS buffer at 4° C. for 1-2 h. The activated NCs are then pelletwashed with pH 6.0 buffer to remove un-reacted EDC/NHS and suspended inpH 6.0 buffer. Human Influenza A virus HA protein trimer dissolved in pH6.0 buffer is then added to the resulting NC suspension. The conjugationis allowed to proceed at 4° C. overnight. After pellet washing with PBSbuffer, the resulting NC-HA protein trimer conjugate is suspended in pH7.4 PBS buffer for further testing.

Streptococcus pneumonia polysaccharide (PnPs) 6B is selected as arepresentative PnPs serotype. Purified native (i.e., no postpurification size reduction) PnPs-6B is dissolved in 2 M NaCl. Asolution of 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP)in CH3CN (100 mg /mL) is added (ratio of CDAP/PnPs: 1.0 mg/mg). The pHof the resulting solution is adjusted to 9 with 0.2 M of aqueous Et3N ordilutes of NaOH solution. After 3-4 min, the resulting activated PnPs-6Bsolution is added to NCs with surface PEG-CONHNH2 (PEG-hydrazide) groupsprepared as described above in Example 13 in pH 9 buffer. The resultingNCs and PnPs-6B suspension is shaken for 1 h and quenched with 2 Mglycine solution. After pellet washing with PBS buffer, the resultingNC-PnPs-6B conjugate is suspended in pH 7.4 PBS buffer.

The nanocarriers can then be combined to form a NC suspension forfurther testing.

Example 19 Monovalent Nanocarriers with Antigens from the Same Strain ofInfectious Agent (Prophetic)

Nanocarriers with PEG-X on the surface are prepared as follows.Monodisperse PRINT nanocarriers (PRINT NCs) comprising PLGA-R848,PLA-PEG-X (where X=carboxylic acid (CO2H), amine (NH2), C6-azide (C6-N3)or PEG3-azide (PEG3-N3), hydrazide (CONHNH2), maleimide (MAL) and thiol(SH)) containing ova peptide are prepared by the Particle Replication inNon-wetting Templates (PRINT) method as described in the literature ((1)“Direct Fabrication and Harvesting of Monodisperse, Shape SpecificNano-Biomaterials”; Rolland, J. P.; Maynor, B. W.; Euliss, L. E.; Exner,A. E.; Denison, G. M.; DeSimone, J. M J. Am. Chem. Soc. 2005, 127,10096; (2) “The Complex Role of Multivalency in Nanoparticles Targetingthe Transferrin Receptor for Cancer Therapies” Jin Wang, Shaomin Tian,Robby A. Petros, Mary E. Napier and Joseph M. DeSimone; J. Am. Chem.Soc., 2010, 132 (32), pp 11306-11313). PRINT-NCs with surface PEG-CO2Hgroups are activated with excess EDC/NHS in pH 6 PBS buffer at 4° C. for1-2 h. The activated NCs are then pellet washed with pH 6.0 buffer toremove un-reacted EDC/NHS and suspended in pH 6.0 buffer. Pneumococcalsurface protein A (PspA) dissolved in pH 6.0 buffer is then added to theresulting NC suspension. The conjugation is allowed to proceed at 4° C.overnight. After pellet washing with PBS buffer, the resulting NC-PsPAconjugate is suspended in pH 7.4 PBS buffer.

Purified native PnPs-6B is dissolved in 2 M NaCl. A solution of1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) in CH3CN (100mg /mL) is added (ratio of CDAP/PnPs: 1.0 mg/mg). The pH of theresulting solution is adjusted to 9 with 0.2 M of aqueous Et3N ordilutes of NaOH solution. After 3-4 min, the resulting activated PnPs-6Bsolution is added to PRINT NCs with surface PEG-CONHNH2 (PEG-hydrazide)groups prepared as described above in pH 9 buffer. The resulting NCs andPnPs-6B suspension is shaken for 1 h and quenched with 2 M glycinesolution. After pellet washing with PBS buffer, the resulting NC-PnPs-6Bconjugates are suspended in pH 7.4 PBS buffer.

The nanocarriers can then be combined to form a NC suspension forfurther testing.

Example 20 Monovalent Nanocarriers with Antigens from Different Strainsof Infectious Agent (Prophetic)

Purified native PnPs-6B is dissolved in 2 M NaCl. A solution of1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) in CH3CN (100mg /mL) is added (ratio of CDAP/PnPs: 1.0 mg/mg). The pH of theresulting solution is adjusted to 9 with 0.2 M of aqueous Et3N ordilutes of NaOH solution. After 3-4 min, the resulting activated PnPs-6Bsolution is added to NCs with surface PEG-CONHNH2 (PEG-hydrazide) groupsprepared as described above in Example 13 in pH 9 buffer. The resultingNCs and PnPs-6B suspension is shaken for 1 h and quenched with 2 Mglycine solution. After pellet washing with PBS buffer, the resultingNC-PnPs-6B conjugates are suspended in pH 7.4 PBS buffer.

Purified native PnPs14 from is dissolved in 2 M NaCl. A solution of1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) in CH3CN (100mg /mL) is added (ratio of CDAP/PnPs: 1.0 mg/mg). The pH of theresulting solution is adjusted to 9 with 0.2 M of aqueous Et3N ordilutes of NaOH solution. After 3-4 min, the resulting activated PnPs14solution is added to PRINT NCs with surface PEG-CONHNH2 (PEG-hydrazide)groups prepared as described above in pH 9 buffer. The resulting PRINTNCs and PnPs14 suspension is shaken for 1 h and quenched with 2 Mglycine solution. After pellet washing with PBS buffer, the resultingPRINT NC-PnPs14 conjugates are suspended in pH 7.4 PBS buffer.

Gold NCs with surface PEG-X (where X=carboxylic acid (CO2H), amine(NH2), azide (N3), hydrazide (CONHNH2) and aldehyde (CHO)) are preparedas follows.

Step-1. Formation of Gold NCs (AuNCs): A aq. solution of 500 mL of 1 mMHAuCl4 is heated to reflux for 10 min with vigorous stirring in a 1 Lround-bottom flask equipped with a condenser. A solution of 50 mL of 40mM of trisodium citrate is then rapidly added to the stirring solution.The resulting deep wine red solution is kept at reflux for 25-30 min andthe heat is withdrawn and the solution is cooled to room temperature.The solution is then filtered through a 0.8 μm membrane filter to givethe AuNCs solution. The AuNCs are characterized using visiblespectroscopy and transmission electron microscopy. The AuNCs are ca. 20nm diameter capped by citrate with peak absorption at 520 nm.

Step-2. AuNCs functionalized with PEG-X using HS-PEG-X: AuNCs arefunctionalized with HS-PEG-X (MW range: 1500-5000) (where X=carboxylicacid (CO2H), amine (NH2), azide (N3), hydrazide (CONHNH2) and aldehyde(CHO)) as follows. A solution of 150 μl of HS-PEG-X (10 μM in 10 mM pH9.0 carbonate buffer) is added to 1 mL of 20 nm diameter citrate-cappedgold nanocarriers (1.16 nM) to produce a molar ratio of thiol to gold of2500:1. The mixture is stirred at room temperature under argon for 1hour to allow complete exchange of thiol with citrate on the goldnanocarriers. The AuNCs with PEG-X on the surface is then purified bycentrifuge at 12,000g for 30 minutes. The supernatant is decanted andthe pellet containing AuNC-PEG-X is re-suspended in appropriate PBSbuffer for further bioconjugation with biomolecules. Purified nativePnPs-19F from is dissolved in 2 M NaCl. A solution of1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) in CH3CN (100mg /mL) is added (ratio of CDAP/PnPs: 1.0 mg/mg). The pH of theresulting solution is adjusted to 9 with 0.2 M of aqueous Et3N ordilutes of NaOH solution. After 3-4 min, the resulting activatedPnPs-19F solution is added to AuNCs with surface PEG-CONHNH2(PEG-hydrazide) groups prepared as described above in pH 9 buffer. Theresulting AuNCs and PnPs-19F suspension is shaken for 1 h and quenchedwith 2 M glycine solution. After pellet washing with PBS buffer, theresulting AuNC-PnPs-19F conjugates are suspended in pH 7.4 PBS buffer.

The nanocarriers can then be combined to form a NC suspension forfurther testing.

Example 21 Monovalent Nanocarriers with the Same Antigen but DifferentOrientation (Prophetic)

PRINT NCs with surface nicotine analog attached via the 3′-position areprepared from PLA-PEG-3-HO-MeNic copolymer derived fromtrans-3′-hydroxymethylnicotine (3-HO-MeNic), PLGA-R848 and ova peptideas described above. The resulting PRINT NCs containing surface3′-substituted nicotine analog are suspended in pH 7.4 buffer.

In a similar manner, PRINT NCs with surface nicotine analog attached viathe 1′-position are prepared from PLA-PEG-1-butyl-Nic copolymer derivedfrom 1′-butyl nicotine (1-butyl-Nic), PLGA-R848 and ova peptide asdescribed above. The resulting PRINT NCs containing surface1′-substituted nicotine analog are suspended in pH 7.4 buffer.

The nanocarriers can then be combined to form a NC suspension forfurther testing.

Example 22 Monovalent Nanocarriers with the Same Antigen but DifferentConformation (Prophetic)

Nanocarriers with surface PEG-CO2H groups are prepared as describedabove in Example 13. The NCs are then activated with excess EDC/NHS inpH 6 PBS buffer at 4° C. for 1-2 h. The activated NCs are then pelletwashed with pH 6.0 buffer to remove un-reacted EDC/NHS and suspended inpH 6.0 buffer. Measles virus hemagglutinin noose epitope (HNE, H379-410,disulfide intact) dissolved in pH 6.0 buffer is then added to theresulting NC suspension. The conjugation is allowed to proceed at 4° C.overnight. After pellet washing with PBS buffer, the resulting NC-HNEconjugates are suspended in pH 7.4 PBS buffer.

The highly conserved hemagglutinin noose epitope (HNE, H379-410) of themeasles virus contains three cysteine residues, two of which (Cys386 andCys394) form a disulfide bridge. The HNE peptide containing thedisulfide bridge is reduced using dithiothreitol (DTT) in PBS buffer togive reduced HNE. NCs with surface PEG-CO2H groups are prepared asdescribed above in Example 13. The NCs are then activated with excessEDC/NHS in pH 6 PBS buffer at 4° C. for 1-2 h. The activated NCs arethen pellet washed with pH 6.0 buffer to remove un-reacted EDC/NHS andsuspended in pH 6.0 buffer. The reduced HNE dissolved in pH 6.0 bufferis then added to the resulting NC suspension under argon in the presenceof DTT. The conjugation is allowed to proceed at 4° C. overnight underargon. After pellet washing with PBS buffer, the resulting NC-reducedHNE conjugates are suspended in pH 7.4 PBS buffer.

The nanocarriers can then be combined to form a NC suspension forfurther testing.

Example 23 Monovalent and Bivalent Nanocarriers with the Small MoleculeAntigens of Different Structure (Prophetic)

AuNCs with surface PEG-CO2H groups are prepared as described above. TheAuNCs are then activated with excess EDC/NHS in pH 6 PBS buffer at 4° C.for 1-2 h. The activated NCs are then pellet washed with pH 6.0 bufferto remove un-reacted EDC/NHS and suspended in pH 6.0 buffer.Trans-3′-aminomethylnicotine prepared from commercially available4-cotininecarboxylic acid (US Patent Application: US2007/0129551 A1) inpH 6.0 buffer is added to the activated AuNCs. The conjugation isallowed to proceed at 4° C. overnight. After pellet washing with PBSbuffer, the resulting AuNC-nicotine conjugates are suspended in pH 7.4PBS buffer.

VLPs with surface functional groups such as azide or alkyne for CuAACclick chemistry are prepared as described in the literature (“SurfaceFunctionalization of Virus-Like Particles by Direct Conjugation UsingAzide—Alkyne Click Chemistry”, Kedar G. Patel and James R. Swartz;Bioconjugate Chem., 2011, 22 (3), pp 376-387). Cocaine analog containingalkyne or azide linker and methamphetamine analog containing alkyne orazide linker are prepared according to literature procedures as surfaceB-cell antigen epitopes. An equal molar mixture of cocaine analog andmethamphetamine analog with azide linker is treated with VLPs containingsurface alkyne group under standard CuAAC condition to giveVLP-cocaine-methamphetamine conjugates.

The nanocarriers can then be combined to form a NC suspension forfurther testing.

Example 24 Bivalent Nanocarriers with Oligosaccharide Antigens ofDifferent Structure (Prophetic)

Purified PnPs-6B is size reduced with dilute acid or under sonication togive oligomeric PnPs-6B which is dissolved in 2 M NaCl. Similarly,purified PnPs-3 is size reduced with dilute acid or under sonication togive oligomeric PnPs-3 which is dissolved in 2 M NaCl. An equal molarmixed solution of oligomeric PnPs-6B and PnPs-3 is prepared from thesesolutions. A solution of 1-cyano-4-dimethylaminopyridiniumtetrafluoroborate (CDAP) in CH3CN (100 mg /mL) is added (ratio ofCDAP/PnPs: 1.5 mg/mg) to the mixed PnPs solution. The pH of theresulting solution is adjusted to 9 with 0.2 M of aqueous Et3N ordilutes of NaOH solution. After 3-4 min, the resulting activatedoligomeric PnPs-6B/PnPs-3 solution is added to AuNCs with surfacePEG-CONHNH2 (PEG-hydrazide) groups prepared as described above in pH 9buffer. The resulting AuNCs and activated PnPs-6B/PnPs-3 suspension isshaken for 1 h and quenched with 2 M glycine solution. After pelletwashing with PBS buffer, the resulting AuNC-PnPs-6B/3 conjugates aresuspended in pH 7.4 PBS buffer.

VLPs containing carboxylic acid (CO2H) groups on the surface areactivated with excess EDC/NHS in pH 6 PBS buffer at 4° C. for 1-2 h. Theactivated VLPs are then pellet washed with pH 6.0 buffer to removeun-reacted EDC/NHS and suspended in pH 6.0 buffer. Purified PnPs-4 issize reduced with dilute acid or under sonication to give oligomericPnPs-4 which is dissolved in 2 M NaCl. Similarly, purified PnPs-19F issize reduced with dilute acid or under sonication to give oligomericPnPs-19F which is dissolved in 2 M NaCl. An equal molar mixed solutionof oligomeric PnPs-4 and PnPs-19F is prepared from these solutions. Asolution of 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP)in CH3CN (100 mg/mL) is added (ratio of CDAP/PnPs: 1.5 mg/mg) to themixed PnPs solution. The pH of the resulting solution is adjusted to 9with 0.2 M of aqueous Et3N or dilutes of NaOH solution. After 3-4 min, asolution of adipic acid dihydrazide (ADH) linker in pH 9 buffer is addedto the activated mixed PnPs-4/19F solution. The resulting solution ismixed for 1 h and quenched with 2 M glycine solution and purified bydialysis. The purified oligomeric PnPs-4/19F with ADH linker in pH 6.0buffer is then added to the activated VLPs in pH 6.0 buffer and theresulting suspension is mixed at 4° C. overnight and purified bydialysis or pellet wash to give VLP-PnPs-4/19F conjugates for furthertesting.

The nanocarriers can then be combined to form a NC suspension forfurther testing.

Example 25 Bivalent Nanocarriers with Polysaccharide Antigens ofDifferent Structure (Prophetic)

Purified native PnPs-6B is dissolved in 2 M NaCl. A solution of1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) in CH3CN (100mg /mL) is added (ratio of CDAP/PnPs: 1.0 mg/mg). The pH of theresulting solution is adjusted to 9 with 0.2 M of aqueous Et3N ordilutes of NaOH solution. After 3-4 min, a solution of adipic aciddihydrazide (ADH) linker in pH 9 buffer is added to the activatedPnPs-6B solution. The resulting solution is mixed for 1 h and purifiedby dialysis. The purified PnPs-6B with ADH linker is dissolved in pH 6.0buffer for NC conjugation.

Purified N. meningitidis meningococcal polysaccharide serogroup A (NmA)is dissolved in 1 M NaCl. A solution of1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) in CH3CN (100mg /mL) is added (ratio of CDAP/NmA: 1.5 mg/mg). The pH of the resultingsolution is adjusted to 9 with 0.2 M of aqueous Et3N or dilutes of NaOHsolution. After 3-4 min, a solution of adipic acid dihydrazide (ADH)linker in pH 9 buffer is added to the activated NmA solution. Theresulting solution is mixed for 1-2 h and purified by dialysis. Thepurified NmA with ADH linker is dissolved in pH 6.0 buffer for NCconjugation.

NCs with surface PEG-CO2H groups are prepared as described above inExample 13. The NCs are then activated with excess EDC/NHS in pH 6 PBSbuffer at 4° C. for 1-2 h. The activated NCs are then pellet washed withpH 6.0 buffer to remove un-reacted EDC/NHS and suspended in pH 6.0buffer. An equal molar mixed solution of PnPs-6B with ADH linker and NmAwith ADH linker in pH 6.0 buffer is added to the activated NC solution,and the resulting suspension is mixed at 4° C. overnight. After pelletwashing with PBS buffer, the resulting NC-PnPs6B/NmA conjugates aresuspended in pH 7.4 PBS buffer.

Purified native PnPs-19F is dissolved in 2 M NaCl. A solution of1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) in CH3CN (100mg /mL) is added (ratio of CDAP/PnPs: 1.0 mg/mg). The pH of theresulting solution is adjusted to 9 with 0.2 M of aqueous Et3N ordilutes of NaOH solution. After 3-4 min, a solution of adipic aciddihydrazide (ADH) linker in pH 9 buffer is added to the activatedPnPs-19F solution. The resulting solution is mixed for 1 h and purifiedby dialysis. The purified PnPs-19F with ADH linker is dissolved in pH6.0 buffer for NC conjugation.

Purified N. meningitidis meningococcal polysaccharide serogroup C (NmC)is dissolved in 1 M NaCl. A solution of1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) in CH3CN (100mg /mL) is added (ratio of CDAP/NmC: 1.5 mg/mg). The pH of the resultingsolution is adjusted to 9 with 0.2 M of aqueous Et3N or dilutes of NaOHsolution. After 3-4 min, a solution of adipic acid dihydrazide (ADH)linker in pH 9 buffer is added to the activated NmC solution. Theresulting solution is mixed for 1-2 h and purified by dialysis. Thepurified NmC with ADH linker is dissolved in pH 6.0 buffer for NCconjugation.

NCs with surface PEG-CO2H groups are prepared as described above inExample 13. The NCs are then activated with excess EDC/NHS in pH 6 PBSbuffer at 4° C. for 1-2 h. The activated NCs are then pellet washed withpH 6.0 buffer to remove un-reacted EDC/NHS and suspended in pH 6.0buffer. An equal molar mixed solution of PnPs-19F with ADH linker andNmC with ADH linker in pH 6.0 buffer is added to the activated NCsolution, and the resulting suspension is mixed at 4° C. overnight.After pellet washing with PBS buffer, the resulting NC-PnPs-19F/NmCconjugates are suspended in pH 7.4 PBS buffer.

The nanocarriers can then be combined to form a NC suspension forfurther testing.

Example 26 Bivalent and Monovalent Nanocarriers with Small MoleculeAntigens in Different Orientations (Prophetic)

Nanocarriers with surface PEG-CONHNH2 (PEG-hydrazide) are prepared asdescribed above in Example 13 and suspended in pH 6.0 buffer at 4° C. Acocaine analog GNC (6-(2R,3S)-3-(benzoyloxy)-8-methyl-8-azabicyclo[3.2.11 octane-2-carbonyloxy-hexanoic acid) is prepared according to areported procedure (“Cocaine Analog Coupled to Disrupted Adenovirus: AVaccine Strategy to Evoke High-titer Immunity Against Addictive Drugs”Martin J Hicks, et al, Mol Ther 2011, 19: 612-619). This compound isactivated with EDC/NHS in DMF, and the activated GNC-NHS ester isisolated and purified for NC conjugation. Another cocaine analog, AI1 isprepared according to a reported procedure (“Positional linker effectsin haptens for cocaine immunopharmacotherapy”, Akira Ino, Tobin J.Dickerson, and Kim D. Janda; Bioorganic & Medicinal Chemistry Letters 17(2007) 4280-4283) and activated with EDC/NHS as above. An equal molarportion of each activated cocaine analog in excess to NC surfacePEG-hydrazide is mixed with the NCs in pH 6.0 buffer. The resultingsuspension is mixed at 4° C. overnight. After pellet washing with PBSbuffer, the resulting NC-GNC/AI1 cocaine conjugates are suspended in pH7.4 PBS buffer.

Norcocaine is treated with succinic anhydride to give cocaine containinga succinic acid linker, SNC, and then activated with EDC/NHS accordingto a reported procedure (Fox B S, Kantak K M, Edwards M A et al.Efficacy of a therapeutic cocaine vaccine in rodent models. Nat. Med.2(10), 1129-1132 (1996). NCs with surface PEG-CONHNH2 (PEG-hydrazide)are prepared as described above in Example 13 and suspended in pH 6.0buffer at 4° C. Excess amounts of activated cocaine analog, SNC, isadded to the NCs. The resulting suspension is mixed at 4° C. overnight.After pellet washing with PBS buffer, the resulting NC-SNC cocaineconjugates are suspended in pH 7.4 PBS buffer.

The nanocarriers can then be combined to form a NC suspension forfurther testing.

Example 27 Bivalent and Monovalent Nanocarriers with Peptide Antigenswith Different Attachments (Prophetic)

Nanocarriers with surface PEG-azide (PEG-N3) are prepared according toExample 13 and suspended in de-gassed pH 7 buffer with argon. Ovalbumin(325-336) peptide with a C-terminal propargyl amide group (a C-alkynegroup) is prepared by standard solid phase peptide synthesis, and theresulting purified Ova (325-336)-C-alkyne peptide is dissolved in pH 7buffer under argon. Ovalbumin (325-336) peptide with the N-terminalamine acylated with 5-hexynoic acid (an N-terminal alkyne group) isprepared by standard solid phase peptide synthesis, and the resultingpurified Ova (325-336)-N-alkyne peptide is dissolved in pH 7 bufferunder argon. NCs with surface PEG-N3is mixed with an equal molar amountof each ova —C-alkyne and N-alkyne peptide in pH 7 buffer under argon,and the resulting suspension is subjected to the CuAAC click reactionaccording to a reported protocol (“Analysis and optimization ofcopper-catalyzed azide-alkyne cycloaddition for bioconjugation”, Hong V,Presolski S I, Ma C, Finn M G.; Angew Chem Int Ed Engl.2009;48(52):9879-83). The resulting NC-Ova peptide-C-linked/Ovapeptide-N-linked conjugates are purified by pellet wash with pH 7 bufferand suspended in pH 7 buffer.

Recombinant virus-like particles (VLP) are prepared according to astandard procedure. In particular, VLPs from rabbit hemorrhagic diseasevirus is prepared and conjugated with Ova (323-339) peptide via aheterobifunctional linker such as Sulfosuccinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate (Sulfo-SMCC) asdescribed by Matthew Peacey, et al. ((1) Peacey M, Wilson S, Baird M A,Ward V K. “Versatile RHDV virus-like particles: incorporation ofantigens by genetic modification and chemical conjugation” BiotechnolBioeng; 2007; 98:968-77; (2) Peacey M, Wilson S, Perret R, Ronchese F,Ward V K, Young V, Young S, Baird, M A. “Virus-like particles fromrabbit hemorrhagic disease virus can induce ananti-tumor response”Vaccine; 2008; 26:5334-5337). The resulting VLP-ova peptide conjugatesare purified and suspended in pH 7 buffer.

The nanocarriers can then be combined to form a NC suspension forfurther testing.

Example 28 Monovalent Nanocarriers with Protein Antigens Coupled atDifferent Attachment Points on the Protein (Activated Versus ProteinTag) (Prophetic)

Nanocarriers with surface PEG-CO2H groups are prepared as described inExample 13. The NCs are then activated with excess EDC/NHS in pH 6 PBSbuffer at 4° C. for 1-2 h. The activated NCs are then pellet washed withpH 6.0 buffer to remove un-reacted EDC/NHS and suspended in pH 6.0buffer. Measles hemaglutinin protein (MHP) dissolved in pH 6.0 buffer isthen added to the resulting NC suspension. The conjugation is allowed toproceed at 4° C. overnight. After pellet washing with PBS buffer, theresulting NC-MHP conjugate is suspended in pH 7.4 PBS buffer.

Nanocarriers with surface PEG-NTA group for Ni-His tag complexation areprepared as described in Example 13. The NCs are then treated with asolution of NiCl2 in a binding buffer (50 mM phosphate buffer system,300 mM NaCl, 10 mM imidazole, pH 8.0) to form the NCs with surfaceNTA-Ni complex. After pellet washing with PBS buffer, the resulting NCsare suspended in the binding buffer under argon. A solution ofHis6-tagged recombinant measles hemaglutinin protein in the bindingbuffer is added to the NC suspension, and the suspension is incubated at4° C. overnight under argon. The resulting NC-NTA-His6-MHP conjugatesare pellet washed with pH 7 buffer and suspended in PBS buffer.

The nanocarriers can then be combined to form a NC suspension forfurther testing.

Example 29 Monovalent Nanocarriers with Oligosaccharide Antigens Coupledat Different Attachment Points (Activated Hydroxyl Group Versus Linker)(Prophetic)

Nanocarriers with surface PEG-CONHNH2 (PEG-hydrazide) groups areprepared as described in Example 13 and suspended in pH 9 buffer underargon. Purified PnPs-6B is size reduced with dilute acid or undersonication to give oligomeric PnPs-6B which is dissolved in 2 M NaCl. Asolution of 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP)in CH3CN (100 mg /mL) is added (ratio of CDAP/PnPs: 1.5 mg/mg) to thePnPs-6B solution. The pH of the resulting solution is adjusted to 9 with0.2 M of aqueous Et3N or dilutes of NaOH solution. After 3-4 min, theresulting activated oligomeric PnPs-6B solution is added to the NCs withsurface PEG-CONHNH2 (PEG-hydrazide) groups. The resulting NCs andactivated PnPs-6B suspension is shaken for 1 h and quenched with 2 Mglycine solution. After pellet washing with PBS buffer, the resultingNC-PnPs-6B conjugates are suspended in pH 7.4 PBS buffer.

Nanocarriers with surface PEG-CO2H groups are prepared as described inExample 13. The NCs are then activated with excess EDC/NHS in pH 6 PBSbuffer at 4° C. for 1-2 h. The activated NCs are then pellet washed withpH 6.0 buffer to remove un-reacted EDC/NHS and suspended in pH 6.0buffer. Oligo PnPs-6B with a 3-aminopropyl linker is prepared accordingto a reported method (“Synthetic 6B Di-, Tri-, andTetrasaccharide-Protein Conjugates Contain Pneumococcal Type 6A and 6BCommon and 6B-Specific Epitopes That Elicit Protective Antibodies inMice”, Jansen W T M, et al. Infect Immun. 2001; 69(2): 787-793). Theoligomeric PnPs-6B-3-propylamine in pH 6 buffer is added to theactivated NCs. The resulting suspension is mixed at 4° C. overnightunder argon. After pellet washing with PBS buffer, the NC-PnPs-6Bconjugates are suspended in pH 7.4 PBS buffer.

The nanocarriers can then be combined to form a NC suspension forfurther testing.

Example 30 Monovalent Nanocarriers with Polysaccharide Antigens Coupledat Different Attachment Points on the Polysaccharide (Prophetic)

NmA is attached via CDAP activated hydroxyl groups to NCs with multipleattachment points. Purified N. meningitidis meningococcal polysaccharideserogroup A (NmA) is dissolved in 1 M NaCl. A solution of1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) in CH3CN (100mg/mL) is added (ratio of CDAP/NmA: 1.5 mg/mg). The pH of the resultingsolution is adjusted to 9 with 0.2 M of aqueous Et3N or dilutes of NaOHsolution. After 3-4 min, a solution of adipic acid dihydrazide (ADH)linker in pH 9 buffer is added to the activated NmA solution. Theresulting solution is mixed for 1-2 h and purified by dialysis. Thepurified NmA with ADH linker is dissolved in pH 6.0 buffer for NCconjugation.

NCs with surface PEG-CO2H groups are prepared as described in Example13. The NCs are then activated with excess EDC/NHS in pH 6 PBS buffer at4° C. for 1-2 h. The activated NCs are then pellet washed with pH 6.0buffer to remove un-reacted EDC/NHS and suspended in pH 6.0 buffer. Asolution of NmA with ADH linkers in pH 6.0 buffer is added to theactivated NC solution, and the resulting suspension is mixed at 4° C.overnight. After pellet washing with PBS buffer, the resulting NC-NmAconjugates are suspended in pH 7.4 PBS bufferS.

NmA is attached to NCs via a terminal amino group. NCs with surfacePEG-CO2H groups are prepared as described in Example 13. The NCs arethen activated with excess EDC/NHS in pH 6 PBS buffer at 4° C. for 1-2h. The activated NCs are then pellet washed with pH 6.0 buffer to removeun-reacted EDC/NHS and suspended in pH 6.0 buffer. Purified NmA issubjected to reductive amination with NH4Cl and sodium cyanoborohydride(NaCNBH3) in pH 7 buffer to give the amino-NmA according to a reportedprocedure (“Development and phase 1 clinical testing of a conjugatevaccine against meningococcus A and C”, Costantino P, Viti S, Podda A,Velmonte M A, Nencioni L, Rappuoli R. Vaccine. 1992; 10(10):691-8). Theamino-NmA is then added to the activated NCs suspension, and and theresulting suspension is mixed at 4° C. overnight. After pellet washingwith PBS buffer, the resulting NC-NmA conjugates are suspended in pH 7.4PBS buffer.

The nanocarriers can then be combined to form a NC suspension forfurther testing.

1. A composition comprising: a dosage form comprising: a firstpopulation of synthetic nanocarriers that comprise a first set ofsurface antigens; a second population of synthetic nanocarriers thatcomprise a second set of surface antigens; and a pharmaceuticallyacceptable excipient; wherein the first set of surface antigens and thesecond set of surface antigens are structurally different. 2-3.(canceled)
 4. The composition of claim 1, wherein the first set ofsurface antigens comprise antigens obtained or derived from a firstinfectious genus and the second set of surface antigens compriseantigens obtained or derived from a second infectious genus. 5-9.(canceled)
 10. The composition of claim 1, wherein the first set ofsurface antigens and/or second set of surface antigens comprise antigensthat are obtained or derived from a virus of the Adenoviridae,Picornaviridae, Herpesviridae, Hepadnaviridae, Flaviviridae,Retroviridae, Orthomyxoviridae, Paramyxoviridae, Papillomaviridae,Rhabdoviridae, Togaviridae or Paroviridae family. 11-12. (canceled) 13.The composition of claim 1, wherein the first set of surface antigensand/or second set of surface antigens comprise antigens that areobtained or derived from a bacteria of the Bordetella, Borrelia,Brucella, Campylobacter, Chlamydia and Chlamydophila, Clostridium,Corynebacterium, Enterococcus, Escherichia, Francisella, Haemophilus,Helicobacter, Legionella, Leptospira, Listeria, Mycobacterium,Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella,Staphylococcus, Streptococcus, Treponema Vibrio or Yersinia genus.14-15. (canceled)
 16. The composition of claim 1, wherein the first setof surface antigens and/or second set of surface antigens compriseantigens that are obtained or derived from a fungus of the Candida,Aspergillus, Cryptococcus, Histoplasma, Pneumocystis or Stachybotrysgenus. 17-18. (canceled)
 19. The composition of claim 1, wherein thefirst set of surface antigens and second set of surface antigenscomprise antigens obtained or derived from an abused or addictivesubstance. 20-41. (canceled)
 42. The composition of claim 1, furthercomprising one or more adjuvants.
 43. The composition of claim 42,wherein the first population of synthetic nanocarriers and/or the secondpopulation of synthetic nanocarriers further comprise an adjuvantcoupled to the synthetic nanocarriers.
 44. The composition of claim 42,wherein the first population of synthetic nanocarriers and/or the secondpopulation of synthetic nanocarriers further comprise an adjuvantcoupled to the synthetic nanocarriers and the composition comprises oneor more admixed adjuvants.
 45. The composition of claim 42, wherein eachof the one or more adjuvants comprises a mineral salt, alum, alumcombined with monphosphoryl lipid (MPL) A of Enterobacteria, MPL®(AS04), AS15, a saponin, QS-21,Quil-A, ISCOMs, ISCOMATRIX™, MF59™,Montanide® ISA 51, Montanide® ISA 720, AS02, a liposome or liposomalformulation, AS01, AS15, synthesized or specifically preparedmicroparticles and microcarriers, bacteria-derived outer membranevesicles of N. gonorrheae or Chlamydia trachomatis, chitosan particles,a depot-forming agent, Pluronic® block co-polymers, specificallymodified or prepared peptides, muramyl dipeptide, an aminoalkylglucosaminide 4-phosphate, RC529, a bacterial toxoid, a toxin fragment,an agonist of Toll-Like Receptors 2, 3, 4, 5, 7, 8 or 9, an adeninederivative, immunostimulatory DNA, immunostimulatory RNA, animidazoquinoline amine, an imidazopyridine amine, a 6,7-fusedcycloalkylimidazopyridine amine, a 1,2-bridged imidazoquinoline amine,imiquimod, resiquimod, an agonist for DC surface molecule CD40, a type Iinterferon, poly I:C, a bacterial lipopolysacccharide (LPS), VSV-G,HMGB-1, flagellin or portions or derivatives thereof, animmunostimulatory DNA molecule comprising CpG, proinflammatory stimulireleased from necrotic cells, urate crystals, an activated component ofthe complement cascade, an activated component of immune complexes, acomplement receptor agonist, a cytokine, or a cytokine receptor agonist.46. (canceled)
 47. The composition of claim 43, wherein the adjuvantcoupled to the first population of synthetic nanocarriers and/or theadjuvant coupled to the second population of synthetic nanocarrierscomprises a TLR-2, -3, -4, -7, -8 or -9 agonist.
 48. The composition ofclaim 47, wherein the adjuvant coupled to the first population ofsynthetic nanocarriers and/or the adjuvant coupled to the secondpopulation of synthetic nanocarriers comprises an immunostimulatorynucleic acid, imidazoquinoline, oxoadenine, MPL, imiquimod orresiquimod.
 49. The composition of claim 44, wherein the admixedadjuvant is an immunostimulatory nucleic acid comprising CpG, AS01,AS02, AS04, AS15, QS-21, a saponin, alum or MPL.
 50. The composition ofclaim 1, wherein the first and second populations of syntheticnanocarriers are present in an amount effective to generate an immuneresponse to the first set of surface antigens and the second set ofsurface antigens in a subject. 51-57. (canceled)
 58. The composition ofclaim 1, wherein the first and/or second population of syntheticnanocarriers further comprise a universal T cell antigen coupledthereto. 59-62. (canceled)
 63. A composition comprising: a dosage formcomprising: a first population of synthetic nanocarriers that comprise afirst set of surface antigens; a second population of syntheticnanocarriers that comprise a second set of surface antigens; and apharmaceutically acceptable excipient; wherein the first set of surfaceantigens and the second set of surface antigens are immunologicallydifferent. 64-65. (canceled)
 66. The composition of claim 63, whereinthe first set of surface antigens comprise antigens obtained or derivedfrom a first infectious genus and the second set of surface antigenscomprise antigens obtained or derived from a second infectious genus.67-71. (canceled)
 72. The composition of claim 63, wherein the first setof surface antigens and/or second set of surface antigens compriseantigens that are obtained or derived from a virus of the Adenoviridae,Picornaviridae, Herpesviridae, Hepadnaviridae, Flaviviridae,Retroviridae, Orthomyxoviridae, Paramyxoviridae, Papillomaviridae,Rhabdoviridae, Togaviridae or Paroviridae family. 73-74. (canceled) 75.The composition of claim 63, wherein the first set of surface antigensand/or second set of surface antigens comprise antigens that areobtained or derived from a bacteria of the Bordetella, Borrelia,Brucella, Campylobacter, Chlamydia and Chlamydophila, Clostridium,Corynebacterium, Enterococcus, Escherichia, Francisella, Haemophilus,Helicobacter, Legionella, Leptospira, Listeria, Mycobacterium,Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella,Staphylococcus, Streptococcus, Treponema Vibrio or Yersinia genus.76-77. (canceled)
 78. The composition of claim 63, wherein the first setof surface antigens and/or second set of surface antigens compriseantigens that are obtained or derived from a fungus of the Candida,Aspergillus, Cryptococcus, Histoplasma, Pneumocystis or Stachybotrysgenus. 79-80. (canceled)
 81. The composition of claim 63, furthercomprising one or more adjuvants.
 82. The composition of claim 81,wherein the first population of synthetic nanocarriers and/or the secondpopulation of synthetic nanocarriers further comprise an adjuvantcoupled to the synthetic nanocarriers.
 83. The composition of claim 81,wherein the first population of synthetic nanocarriers and/or the secondpopulation of synthetic nanocarriers further comprise an adjuvantcoupled to the synthetic nanocarriers and the composition comprises oneor more admixed adjuvants.
 84. The composition of claim 81, wherein eachof the one or more adjuvants comprises a mineral salt, alum, alumcombined with monphosphoryl lipid (MPL) A of Enterobacteria, MPL®(AS04), AS15, a saponin, QS-21,Quil-A, ISCOMs, ISCOMATRIX™, MF59™,Montanide® ISA 51, Montanide® ISA 720, AS02, a liposome or liposomalformulation, AS01, synthesized or specifically prepared microparticlesand microcarriers, bacteria-derived outer membrane vesicles of N.gonorrheae or Chlamydia trachomatis, chitosan particles, a depot-formingagent, Pluronic® block co-polymers, specifically modified or preparedpeptides, muramyl dipeptide, an aminoalkyl glucosaminide 4-phosphate,RC529, a bacterial toxoid, a toxin fragment, an agonist of Toll-LikeReceptors 2, 3, 4, 5, 7, 8 or 9, an adenine derivative,immunostimulatory DNA, immunostimulatory RNA, an imidazoquinoline amine,an imidazopyridine amine, a 6,7-fused cycloalkylimidazopyridine amine, a1,2-bridged imidazoquinoline amine, imiquimod, resiquimod, an agonistfor DC surface molecule CD40, a type I interferon, poly I:C, a bacteriallipopolysacccharide (LPS), VSV-G, HMGB-1, flagellin or portions orderivatives thereof, an immunostimulatory DNA molecule comprising CpG,proinflammatory stimuli released from necrotic cells, urate crystals, anactivated component of the complement cascade, an activated component ofimmune complexes, a complement receptor agonist, a cytokine, or acytokine receptor agonist.
 85. (canceled)
 86. The composition of claim82, wherein the adjuvant coupled to the first population of syntheticnanocarriers and/or the adjuvant coupled to the second population ofsynthetic nanocarriers comprises a TLR-2, -3, -4, -7, -8 or -9 agonist.87. The composition of claim 86, wherein the adjuvant coupled to thefirst population of synthetic nanocarriers and/or the adjuvant coupledto the second population of synthetic nanocarriers comprises animmunostimulatory nucleic acid, imidazoquinoline, oxoadenine, MPL,imiquimod or resiquimod.
 88. The composition of claim 83, wherein theadmixed adjuvant is an immunostimulatory nucleic acid comprising CpG,AS01, AS02, AS04, AS15, QS-21, a saponin, alum or MPL.
 89. Thecomposition of claim 63, wherein the first and second populations ofsynthetic nanocarriers are present in an amount effective to generate animmune response to the first set of surface antigens and the second setof surface antigens in a subject. 90-96. (canceled)
 97. The compositionof claim 63, wherein the first and/or second population of syntheticnanocarriers further comprise a universal T cell antigen coupledthereto. 98-102. (canceled)
 103. The composition of claim 1, whereinsynthetic nanocarriers of each of the populations of syntheticnanocarriers comprise lipid-based nanoparticles, polymericnanoparticles, metallic nanoparticles, surfactant-based emulsions,dendrimers, buckyballs, nanowires, virus-like particles, peptide orprotein-based particles, lipid-polymer nanoparticles, spheroidalnanoparticles, cuboidal nanoparticles, pyramidal nanoparticles, oblongnanoparticles, cylindrical nanoparticles, or toroidal nanoparticles.104. The composition of claim 103, wherein each of the populations ofsynthetic nanocarriers comprise one or more polymers. 105-109.(canceled)
 110. A composition comprising: a dosage form comprising: afirst synthetic nanocarrier means for presenting a first set of surfaceantigens; a second synthetic nanocarrier means for presenting a secondset of surface antigens; and a pharmaceutically acceptable excipient;wherein the first set of surface antigens and the second set of surfaceantigens are structurally different.
 111. (canceled)
 112. A compositioncomprising: a dosage form comprising: a first synthetic nanocarriermeans for presenting a first set of surface antigens; a second syntheticnanocarrier means for presenting a second set of surface antigens; and apharmaceutically acceptable excipient; wherein the first set of surfaceantigens and the second set of surface antigens are immunologicallydifferent.
 113. (canceled)
 114. A method comprising: administering thecomposition of claim 1 to a subject.
 115. The method of claim 114,wherein the subject has or is at risk of having an infection orinfectious disease.
 116. The method of claim 114, wherein the subjecthas or is at risk of having cancer.
 117. The method of claim 114,wherein the subject has or is at risk of having an addiction. 118.(canceled)
 119. A method comprising: preparing a first population ofsynthetic nanocarriers that comprise a first set of surface antigens;preparing a second population of synthetic nanocarriers that comprise asecond set of surface antigens; and combining the first and secondpopulations of synthetic nanocarriers into a dosage form; wherein thefirst set of surface antigens and the second set of surface antigens arestructurally different.
 120. A method comprising: preparing a firstpopulation of synthetic nanocarriers that comprise a first set ofsurface antigens; preparing a second population of syntheticnanocarriers that comprise a second set of surface antigens; andcombining the first and second populations of synthetic nanocarriersinto a dosage form; wherein the first set of surface antigens and thesecond set of surface antigens are immunologically different.
 121. Themethod of claim 119, further comprising administering the dosage form toa subject. 122-124. (canceled)
 125. A process for producing a dosageform of a composition, the process comprising the method steps asdefined in claim
 119. 126-132. (canceled)